US6022853A - Morphogen-enriched dietary composition - Google Patents

Morphogen-enriched dietary composition Download PDF

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US6022853A
US6022853A US08/278,730 US27873094A US6022853A US 6022853 A US6022853 A US 6022853A US 27873094 A US27873094 A US 27873094A US 6022853 A US6022853 A US 6022853A
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morphogen
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Thangavel Kuberasampath
Charles M. Cohen
David C. Rueger
Hermann Oppermann
Roy H. L. Pang
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Creative Biomolecules Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention relates generally to the field of dietary compositions and supplements.
  • the present invention relates to compositions useful as mammalian dietary compositions and supplements.
  • the invention relates to food additives and dietary supplements capable of enhancing tissue morphogenesis and development, particularly in individuals at risk for normal tissue development and viability.
  • individuals include infants, particularly prematurely-born ("preterm") and low birth weight infants, and juveniles; aged individuals; and individuals experiencing altered metabolic function and/or suffering from metabolic dysfunctions and other disorders that threaten organ or tissue function or viability, such as can result from malnutrition or starvation, autoimmune diseases, organ cirrhosis and other tissue necrotizing dysfunctions, or disorders associated with aging cells (cell senescence.)
  • Mammalian infants are nourished by mother's milk until such time as they can digest food solids.
  • Infant formulas now exist for humans and other mammals which can supplant or supplement mother's milk.
  • the formulas may be milk based (e.g., cow milk) or non-milk-based (e.g., soy).
  • Particularly at risk are prematurely born infants whose tissues and organs are at an earlier stage of development, and whose nutritional requirements may differ from those of full term infants.
  • Formula development is an ongoing endeavor to more accurately mimic the beneficial aspects of mother's milk. Nevertheless, despite the efforts of many researchers, infant formulas still differ in a number of significant ways from human milk.
  • human milk has many substances, such as immunoglobulins, free amino acids, polyamines, nucleotides and polyunsaturated fatty acids not present, for example, in cow's milk.
  • infant formulas try to mimic the protein quantity found in human milk
  • the foreign proteins typically are present in the formula as hydrolysates to avoid rejection or reaction by the infant's digestive system.
  • the proteins are present primarily as amino acid sources rather than as functional proteins as might normally be transmitted by the nursing mother to the infant.
  • human milk may contain unidentified growth and differentiation factors that are important for overall tissue and skeletal development.
  • Another group of individuals with potentially unique nutritional requirements are individuals undergoing metabolic changes which may result from periods of intense growth or stress, including, for example, pregnant women and drowning victims. Other sources of stress to the body may result from malnutrition or starvation, or from metabolic disorders that affect organ viability, such as autoimmune disease and organ cirrhosis. Aged individuals, and postmenopausal women also have altered or slower metabolic function. All of these individuals are at risk for tissue damage or loss of tissue function due to altered metabolic function.
  • the present invention provides compositions and methods useful in dietary applications and capable of enhancing tissue morphogenesis, including tissue growth, development, maintenance and viability in a mammal, particularly a human.
  • the dietary compositions and supplements of this invention comprise a morphogenic protein ("morphogen"), as described herein, which, when provided to an individual as a food formulation or supplement, is capable of enhancing tissue development, growth, maintenance and/or viability in the individual.
  • morphogen morphogenic protein
  • the compositions and processes provided herein are suitable for both infants and adults, and as part of clinical nutrition.
  • enhancing tissue viability is understood to mean protecting tissue from lost or reduced tissue function due to cell damage or cell senescence, including inducing cells to maintain their differentiated phenotype, inducing regeneration of damaged tissue, and/or inhibiting additional damage thereto.
  • “Morphogenically effective concentration” is understood to mean a concentration sufficient to enhance tissue development and tissue viability in an individual at risk for tissue damage and/or reduced or lost tissue function due to insufficient nutritional considerations, tissue damage associated therewith, and/or incomplete tissue development, regardless of etiology.
  • tissue of the gastrointestinal tract including the oral mucosa, liver tissue, dentin tissue, periodontal tissue, nerve tissue, bone tissue, and any tissue at risk of damage due to immune response-mediated tissue destruction, including ischemia-reperfusion related tissue damage are disposed in copending U.S. Ser. No. 07/945,286 filed Sep. 15, 1992, (currently pending as U.S. Ser. No. 08/461,397), U.S. Ser. No. 07/946,238, filed Sep. 16, 1992 (currently pending as U.S. Ser. No. 08/445,468), U.S. Ser. No. 07/752,764, filed Aug.
  • “Morphogen-solubilizing molecule” is understood to mean a molecule capable of maintaining a morphogen in soluble form in physiologically buffered solutions.
  • Food formulation is understood to mean a dietary composition normally ingested by an individual to satisfy the body's fundamental nutritional requirements;
  • dietary supplement is understood to mean supplemental compositions ingested by an individual in addition to the food formulations ingested to satisfy the fundamental nutritional requirements.
  • Multivitamin and iron tablets are examples of common dietary supplements.
  • Dietary composition is understood to include both food formulations and dietary supplements.
  • infant formula is understood to refer to the well established infant compositions as defined by the American Academy of Pediatrics (AAP) and the AAP Committee on Nutrition ((1985) Pediatrics 75: 976, the European Society of Pediatric Gastroenterology and Nutrition (ESPGAN) and the ESPGAN Committee on Nutrition ((1987) Acta Paed Scan Suppl: 330), including recent updates published by these committees on infant formula nutritional guidelines.
  • the dietary composition or supplement preferably is administered orally, and may be provided in liquid form or as a powder to be dissolved in a beverage.
  • the dietary supplement may be provided as a solid, e.g., in a capsular, tablet, troche or lozenge form; or, the supplement may be provided as an aerosol, for oral or nasal administration.
  • oral administration is not possible or desirable, other administration routes are envisioned.
  • parenteral administration may be required, e.g., via an enteral feeding tube.
  • the morphogen may be provided alone or in association with one or more suitable excipients or carriers, and/or in combination with other beneficial molecules such as vitamins, minerals, lipids, fiber sources and the like.
  • the dietary supplements also may include pharmaceutically acceptable inert materials for use as binders or stabilizers, including magnesium stearate or calcium carbonate.
  • the morphogen may be formulated together with one or more normal food ingredients, e.g., as part of a food formulation. Alternatively or, in addition, the morphogen may be provided as a dietary supplement in, for example, tablet or syrup form.
  • a morphogen precursor "pro" domain which is known to enhance the solubility of the protein in physiologically buffered solutions.
  • Other useful molecules known to enhance protein solubility include casein, including derivatives, salts and analogs thereof, as well as other milk components, and various serum and milk serum proteins.
  • Additional useful molecules which may be associated with the morphogen include tissue targeting molecules capable of directing the morphogen to a desired target tissue.
  • Tissue targeting molecules envisioned to be useful in the treatment protocols of this invention include antibodies, antibody fragments or other binding proteins which interact specifically with surface molecules on the target tissue cells.
  • Still another useful tissue targeting molecule may be part or all of a morphogen precursor "pro" domain.
  • Morphogens may be synthesized in one tissue and secreted and transported to another tissue.
  • the primary source of OP-1 synthesis appears to be the tissue of the urogenic system (e.g., renal and bladder tissue), with secondary expression levels occurring in the brain, heart, lungs and gastrointestinal tract (GI tract, see below.)
  • the protein has been identified in serum, saliva and various milk forms.
  • the secreted form of the protein comprises the mature dimer in association with the pro domain of the intact morphogen sequence.
  • the associated morphogen pro domains may act to target specific morphogens to different tissues in vivo.
  • morphogen species comprising the pro domain may be obtained from the culture medium of morphogen-secreting mammalian cells.
  • a tissue-targeting species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • Associated tissue targeting or solubility-enhancing molecules also may be covalently linked to the morphogen using standard chemical means.
  • the morphogen comprises part of an infant formula.
  • the infant formula may be milk-based or nonmilk-based, e.g., soy-based.
  • a typical ready-to-feed morphogen-enriched formulation for infants when diluted to feeding concentrations, comprises, in addition to the morphogen added to the formula, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.
  • the dietary composition is formulated for individuals at risk for reduced or lost tissue function, such as postmenopausal women, elderly individuals, undernourished or malnourished individuals, dehydrated individuals, drowning victims, individuals suffering from metabolic disorders including an endocrine imbalance, gastrointestinal disorders, or immune-compromised individuals.
  • Undernourished or malnourished individuals include those suffering from a lack of food (starvation) and/or eating disorders (e.g., anorexia nervosa), and/or suffering from a maladsorption syndrome (e.g., individuals afflicted with digestive or intestinal fistulas, shortened bowel, or hypercatabolism.)
  • a medical therapy including radiotherapy, chemotherapy or a surgical procedure also are at risk for reduced or lost tissue function as a result of a therapy-related malabsorption-malnutrition dysfunction.
  • the dietary supplement is formulated for individuals undergoing periods of increased growth or stress, such as infants and juveniles, or pregnant or lactating women.
  • the dietary supplement is formulated for individuals at risk for reduced or lost organ function as results from tissue cirrhosis or an autoimmune disease.
  • Morphogen-enriched nutritional products particularly clinical nutrition products for use in hospital or other clinical settings, in addition to comprising a morphogen preferably are based on the utilization of diverse other protein sources (casein, sodium and calcium caseinate, isolated soy protein, protein hydrolyzates and/or crystalline amino acids) mixtures of vegetable and animal fats, carbohydrates (basically glucose polymers), vitamins and minerals to meet, at least, the dietary intakes recommended for healthy individuals (see, for example, Committee on Dietary Allowances, Food and Nutrition Board, Nat Acad Sci, 9th Ed, 1980).
  • protein sources casein, sodium and calcium caseinate, isolated soy protein, protein hydrolyzates and/or crystalline amino acids
  • carbohydrates basic glucose polymers
  • vitamins and minerals to meet, at least, the dietary intakes recommended for healthy individuals (see, for example, Committee on Dietary Allowances, Food and Nutrition Board, Nat Acad Sci, 9th Ed, 1980).
  • morphogens useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, see Lee (1991) PNAS 88: 4250-4254), all of which are presented in Table II and Seq. ID Nos.5-14), and the recently identified 60A protein (from Drosophila, Seq. ID No. 24, see Wharton et al.
  • DPP from Drosophila
  • Vgl from Xenopus
  • Vgr-1 from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.
  • GDF-1 from mouse, see Lee (1991) PNAS 88: 4250-4254
  • 60A protein from Drosophila
  • the members of this family which include members of the TGF- ⁇ super-family of proteins, share substantial amino acid sequence homology in their C-terminal regions.
  • the proteins are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature sequence.
  • the "pro" form of the protein includes the pro domain and the mature domain, and forms a soluble species that appears to be the primary form secreted from cultured mammalian cells.
  • the signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14: 4683-4691.)
  • Table I below, describes the various morphogens identified to date, including their nomenclature as used herein, their Seq. ID references, and publication sources for the amino acid sequences for the full length proteins not included in the Seq. Listing. The disclosure of these publications is incorporated herein by reference.
  • the OP-2 proteins have an additional cysteine residue in this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in addition to the conserved cysteine skeleton in common with the other proteins in this family.
  • the GDF-1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq. ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded structure.
  • the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton.
  • a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No.
  • cysteines e.g., amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure
  • the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein.
  • the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells.
  • these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.
  • the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); where each Xaa indicates one of the 20 naturally-occurring L-isomer, ⁇ -amino acids or a derivative thereof.
  • Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine identified in OP-2 (see residue 36, Seq. ID No. 2).
  • these sequences further comprise the following additional sequence at their N-terminus:
  • Generic Sequence 3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31), listed below.
  • Generic Sequences 3 and 4 are composite amino acid sequences of the following proteins presented in Table II and identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq.
  • Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among all the morphogen protein family members identified in Table II.
  • Generic Sequences 5 and 6 are composite amino acid sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No.
  • Vgl from Xenopus, Seq. ID No. 12
  • Vgr-1 from mouse, Seq. ID No. 13
  • GDF-1 from mouse, Seq. ID No. 14
  • human BMP3 Seq. ID No. 26
  • human BMP5 Seq. ID No. 27
  • human BMP6 Seq. ID No. 28
  • 60(A) from Drosophila, Seq. ID Nos. 24-25.
  • the generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 5 and 6, respectively), as well as alternative residues for the variable positions within the sequence.
  • Generic Sequences 5 and 6 allow for an additional cysteine at position 41 (Generic Sequence 5) or position 46 (Generic Sequence 6), providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.
  • Particularly useful sequences for use as morphogens in this invention include the C-terminal domains, e.g., the C-terminal 96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as well as proteins comprising the C-terminal domains of 60A, BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at least the conserved six or seven cysteine skeleton.
  • sequences include the inhibins/activin proteins (see, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other useful sequences are those sharing at least 70% amino acid sequence homology or "similarity", and preferably 80% homology or similarity with any of the sequences above. These are anticipated to include allelic and species variants and mutants, and biosynthetic muteins, as well as novel members of this morphogenic family of proteins.
  • amino acid changes from the preferred sequences include conservative changes, e.g., those as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362, (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington, D.C. 1979).
  • potentially useful sequences are aligned with a known morphogen sequence using the method of Needleman et al. ((1970) J.Mol.Biol. 48: 443-453) and identities calculated by the Align program (DNAstar, Inc.).
  • "Homology” or “similarity” as used herein includes allowed conservative changes as defined by Dayoff et al.
  • the currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in another preferred aspect of the invention, useful morphogens include active proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as "OPX", which accommodates the homologies between the various identified species of OP1 and OP2 (Seq. ID No. 29).
  • OPX generic amino acid sequence
  • the morphogens useful in the methods, composition and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and species variants of these proteins, naturally-occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein.
  • the proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
  • the morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions.
  • Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells.
  • a detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in copending U.S. patent application Ser. No. 752,764, filed Aug. 30, 1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of which are incorporated herein by reference.
  • skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins useful as dietary compositions for enhancing tissue morphogenesis, including enhancing tissue development and tissue viability in a variety of mammals, including humans.
  • FIG. 1A and B shows relative amounts of protein present in mammary gland extract eluate fractions of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot (B);
  • FIG. 2A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme A of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western blot under reduced (1) and oxidized (2) conditions (B);
  • FIG. 3A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme B of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot under reduced conditions (B);
  • FIG. 4A and B shows relative amounts of protein present in bovine 57 day milk eluate fractions of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot under reduced (1) and oxidized (2) conditions (B);
  • FIG. 5 shows Western Blot analysis of bovine colostrum using OP-1 and BMP2-specific antibodies
  • FIG. 6A and B show results of in vivo and in vitro activity assays, respectively, for the corresponding fractions shown in FIG. 1;
  • FIG. 7 is a photomicrograph of an immunoblot showing the presence of hOP-1 in serum.
  • FIG. 8A is a dose response curve for the induction of the 180 kDa and 140 kDa N-CAM isoforms in morphogen-treated NG108-15 cells;
  • FIG. 8B is a photomicrograph of a Western blot of whole cell extracts from morphogen-treated NG108-15 cells with an N-CAM-specific antibody.
  • FIG. 9 are photographs showing the effect of morphogen-specific antibody on mouse development (9B) compared to untreated, control mice (9A).
  • proteins described herein are found in nursing mother's milk and are useful as components of a dietary composition for enhancing tissue morphogenesis in a mammal, particularly in an individual at risk for normal tissue development and viability.
  • these proteins are capable of enhancing tissue development in growing mammals, stimulating CAM expression and maintaining the normal tissue function in adult tissue.
  • Suitable morphogens useful in the compositions and methods of this invention, as well as methods for their administration and application, and numerous, nonlimiting examples which demonstrate the suitability of the morphogens described herein as active components of a dietary composition for a mammal; and 2) provide assays with which to test candidate morphogens for their efficacy.
  • examples are provided which (1) demonstrate the presence of endogenous morphogen in milk and human serum (Examples 1 and 2), (2) demonstrate the ability of morphogens to induce CAM expression in a mammal (Example 3), (3) demonstrate the ability of morphogens to enhance tissue development in developing embryos (Example 4) and juveniles (Example 5); (4) demonstrate the ability of morphogens to reduce an osteoporotic condition in a mammal (Example 6); (5) demonstrate the presence of morphogens in developing tissues and adult stomach and gut tissue, demonstrate the ability of parenterally provided morphogen to localize to stomach tissue, and describe protocols for identifying morphogen-synthesizing tissue (Example 7) and (6) describe protocols for obtaining morphogen-specific antibodies and measuring morphogens in solution (Example 8).
  • a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton or its functional equivalent (see supra).
  • the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells.
  • a candidate morphogen or morphogen composition can be evaluated for in vivo morphogenic utility generally according to the procedures set forth in U.S. Ser. No. 08/404,113.
  • the proteins and compositions may be injected or surgically implanted in a mammal, following any of a number of procedures well known in the art.
  • surgical implant bioassays may be performed essentially following the procedure of Sampath et al. (1983) PNAS 80: 6591-6595.
  • Histological sectioning and staining is preferred to determine the extent of morphogenesis in vivo, particularly in tissue repair procedures.
  • Excised implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 ⁇ m sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of the new tissue. Twelve day implants are usually sufficient to determine whether the implants contain newly induced tissue.
  • tissue morphogenesis In addition to histological evaluation, biological markers may be used as a marker for tissue morphogenesis.
  • Useful markers include tissue-specific enzymes whose activity may be assayed (e.g., spectrophotometrically) after homogenization of the implant. These assays may be useful for quantitation and for obtaining an estimate of tissue formation quickly after the implants are removed from the animal. For example, alkaline phosphatase activity may be used as a marker for osteogenesis.
  • Incorporation of systemically provided morphogens may be followed using tagged morphogens (e.g., radioactively labelled) and determining their localization in new tissue, and/or by monitoring their disappearance from the circulatory system using a standard pulse-chase labeling protocol.
  • the morphogen also may be provided with a tissue-specific molecular tag, whose uptake may be monitored and correlated with the concentration of morphogen provided.
  • tissue-specific molecular tag whose uptake may be monitored and correlated with the concentration of morphogen provided.
  • the morphogen to be assayed according to the above-described exemplary procedures can be purified from naturally-sourced material, or can be recombinantly produced from procaryotic or eucaryotic host cells, into which genetic material encoding a morphogen, e.g., genetic material bearing one of the nucleic acid sequences disclosed herein, has been introduced.
  • the above-described exemplary procedures can be used to determine whether a novel protein suspected of being a morphogen indeed has morphogenic activity.
  • Particularly useful proteins include those which comprise the naturally derived sequences disclosed in Table II.
  • Other useful sequences include biosynthetic constructs such as those disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).
  • morphogens useful in the methods and compositions of this invention also may be described by morphogenically active proteins having amino acid sequences sharing 70% or, preferably, 80% homology (similarity) with any of the sequences described above, where "homology" is as defined herein above.
  • Generic Sequences 1, 2, 3, 4, 5 and 6 The morphogens useful in the method of this invention also can be described by any of the 6 generic sequences described herein (Generic Sequences 1, 2, 3, 4, 5 and 6). Generic sequences 1 and 2 also may include, at their N-terminus, the sequence
  • Table II compares the amino acid sequences of the active regions of native proteins that have been id entified as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq.
  • human OP-1 hOP-1, Seq. ID Nos. 5 and 16-17
  • mouse OP-1 mOP-1, Seq. ID Nos. 6 and 18-19
  • human and mouse OP-2 Seq. ID Nos. 7, 8, and 20-23
  • amino acid residue 60 of CBMP-2A and CBMP-2B is "missing".
  • both these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
  • the currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in still another preferred aspect, the invention includes morphogens comprising species of polypeptide chains having the generic amino acid sequence referred to herein as "OPX", which defines the seven cysteine skeleton and accommodates the identities between the various identified mouse and human OP1 and OP2 proteins. OPX is presented in Seq. ID No.
  • each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 16-23).
  • the morphogens may be provided to an individual by any suitable means, most preferably orally, or, alternatively, parenterally.
  • the morphogen preferably comprises part of an aqueous solution.
  • the solution is physiologically acceptable so that in addition to delivery of the desired morphogen to the patient, the solution does not otherwise adversely affect the patient's electrolyte and volume balance.
  • the aqueous medium for the morphogen thus may comprise normal physiologic saline (0.85% NaCl, 0.15 M), pH 7-7.4.
  • the aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents.
  • TFA trifluoroacetic acid
  • One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA).
  • PBS phosphate buffered saline
  • HSA human serum albumin
  • the resultant solution preferably is vortexed extensively.
  • a given morphogen may be made more soluble by association with a suitable molecule.
  • the pro form of the morphogenic protein comprises a species that is soluble in physiologically buffered solutions.
  • the endogenous protein is thought to be transported (e.g., secreted and circulated) in this form.
  • This soluble form of the protein may be obtained from the culture medium of morphogen-secreting mammalian cells.
  • a soluble species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • Another molecule capable of enhancing solubility and particularly useful for oral administrations is casein, including salts, derivatives and analogs thereof. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 in physiologically buffered solutions by 80%.
  • Other components found in milk and/or various serum proteins also may be useful.
  • Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990.
  • Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Biocompatible, preferably bioresorbable, polymers including, for example, hyaluronic acid, collagen, polybutyrate, tricalcium phosphate, lactide and lactide/glycolide copolymers, may be useful excipients to control the release of the morphogen in vivo.
  • the dietary supplements comprising the morphogens described herein preferably are provided orally.
  • Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream.
  • the morphogens described herein typically are acid stable and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.)
  • at least one morphogen, OP-1 has been identified in mammary gland extract, colostrum and 57-day milk (see Example 1, below).
  • the OP-1 purified from mammary gland extract is morphogenically active.
  • this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen also is detected in the bloodstream (see Example 2, below).
  • the morphogen form found in milk is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.
  • the dietary compositions for oral administration may be formulated as a liquid, for example, as part of an aqueous medium as described above for parenteral administration, and which further may contain flavoring and coloring agents.
  • the formulation also may be combined with a beverage or may be provided in a syrup.
  • the dietary composition also may be provided as an aerosol for oral or nasal administration.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • the dietary composition may be provided as a solid, for example as a tablet, capsule or lozenge.
  • formulations for oral administration also may include molecules to enhance a controlled release of the morphogen in vivo.
  • the concentration of the compounds described in a given dietary supplement composition will vary depending upon a number of factors, including the dosage number to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration.
  • the preferred dosage to be administered also is likely to depend on such variables as the type and extent of tissue development enhancement desired, the type and extent of any tissue damage present to be repaired, the overall health status of the particular individual, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration.
  • the compounds of this invention may be provided in a formulation containing about 0.001 to 10% w/v of morphogen to formulation.
  • Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.1 ⁇ g/kg to 100 mg/kg of body weight per day.
  • the morphogen dosage given is between 0.1-100 ⁇ g of protein per kilogram weight of the individual. No obvious morphogen induced pathological lesions are induced when mature morphogen (e.g., OP-1, 20 ⁇ g) is administered daily to normal growing rats for 21 consecutive days.
  • 10 ⁇ g systemic injections of morphogen (e.g., OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.
  • a large volume loading dose is used at the start of the treatment.
  • the treatment then is continued with a maintenance dose.
  • administration dosages then can be monitored by measuring at intervals the levels of the morphogen in the blood.
  • the morphogens of this invention preferably are added to an infant formula that complies with the nutritional guidelines provided by the AAP and ESPGAN.
  • Basic ingredients for infant formulas include cow's milk, protein, whey proteins, casein and its salts (i.e. calcium caseinate).
  • Soy protein isolates may be substituted for milk-derived proteins, and preferably are used in the products made for infants with lactose intolerance and/or cow's protein intolerance.
  • Protein hydrolyzates i.e. casein and lactalbumin hydrolyzates with low molecular weight, also may be used for these products.
  • the proportions of the diverse component nutrients preferably are similar to those of human milk.
  • the ratio of whey proteins to casein preferably varies from 60:40 to 70:30 in infant formulas based on milk.
  • the mixture of fats employed is made up of edible fats to provide an essential fatty acid profile.
  • Lactose preferably is used as the carbohydrate source for at-term newborns infants, and dextrinmaltose preferably is employed in products used for the treatment of lactose intolerance and malabsorption syndromes in infancy.
  • Infant formulas according to the invention also preferably contain minerals (including calcium, phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese and iodine) and vitamins (including vitamin A, vitamin D3, vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E, vitamin K1, folic acid, biotin) adequate for the infants' requirements.
  • minerals including calcium, phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese and iodine
  • vitamins including vitamin A, vitamin D3, vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E, vitamin K1, folic acid, biotin
  • carnitine preferably is included to satisfy the nutritional requirements for this compound in infants with malabsorptive syndromes.
  • a typical ready-to-feed morphogen-enriched formulation for infants when diluted to feeding concentrations, preferably comprises in addition to the added morphogen, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.
  • a typical protein source for use in infant formula is electrodialyzed whey or electrodialyzed skim milk or milk whey, although other protein sources are also available and may be used.
  • Preferred sugars include food grade substances such as glucose, dextrose, sucrose, or edible lactose.
  • the following vitamins and minerals may also be incorporated in the infant formula: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, and B complex. These micronutrients are added in the form of commonly accepted nutritional compounds in amounts equivalent to those present in human milk on a per calories basis.
  • the infant formula according to the present invention also preferably is sterilized and subsequently used on a ready-to-feed basis, or can be stored as a concentrate.
  • the concentrate can be prepared using standard procedures known in the art, and the formula can be reconstituted by rehydrating the concentrate.
  • the infant formula preferably is a stable liquid and has a suitable shelf life.
  • the morphogen-enriched dietary products for balanced nutrition e.g., dietary food formulations
  • a composition of nutrients adequate to the specific requirements of not only healthy human in need of a balanced nutritional product, but also those individuals at risk for lost or reduced tissue function due malnutrition-maladsorption disorder, and/or altered metabolism.
  • Individuals particularly affected by an altered metabolic function include postmenopausal women or aged individuals, hypercatabolic individuals, and individuals undergoing periods of rapid growth or physical stress, such as developing juveniles, and pregnant, lactating and nursing mothers.
  • Other individuals at risk are those suffering from malnutrition, induced, for example, by starvation and/or an eating disorder, and individuals affected with energy-protein malnutrition and in hypercatablic states derived from traumatic, septic, surgical processes and other clinically-derived malabsorption syndromes.
  • Morphogen-enriched nutritional products preferably also provide mineral elements which include trace elements and vitamins in adequate proportions to satisfy the specific requirements of normal healthy individuals as well as individuals at risk, such as those suffering malabsorption-malnutrition processes and in a hypercatabolic state.
  • the nutritional products also preferably are enriched with amino acids sources, vitamins, nucleosides and/or nucleotides in similar amounts to those present in ordinary foods.
  • liquid products may be formulated ready for consumption or as concentrates to be diluted before use.
  • liquid dietary compositions have pH values generally ranging from about 6.0 to about 8.0, most preferably 6.8-7.5.
  • Morphogenically active OP-1 was demonstrated to be present in mammary gland extract, colostrum, and milk, as described below.
  • the discovery that the morphogen naturally is present in milk together with the known observation that mature, active OP-1 is acid-stable and protease-resistant, indicate that oral administration is a useful route for therapeutic administration of morphogen to a mammal. Oral administration typically is the preferred mode of delivery for extended or prophylactic therapies.
  • the identification of morphogen in all milk forms, including colostrum indicates that the protein plays a significant role in tissue development, including skeletal development of juveniles.
  • Rat mammary gland extract and bovine colostrum and 57 day milk were subjected to purification procedures designed to partially purify OP-1.
  • the partially purified product then was examined for the presence of OP-1 by Western blot analysis using OP-1-specific antisera, and tested for in vivo and in vitro activity.
  • the purification protocol for all three "milk” forms involved three chromatography steps: (1) cation-exchange chromatography (S-Sepharose and followed by Phenyl-Sepharose chromatography); (2) Copper-Immobilized Metal Affinity chromatography (Cu++-IMAC); and finally, (3) C-18 reverse phase chromatography.
  • Fractions were sampled at each step for the presence of OP-1. Fraction samples for testing were dialyzed versus water/0.1% TFA, then against 30% acetonitrile/0.1% TFA for analysis on SDS-polyacrylamide gels and immunoblots, using standard methodologies well described in the art.
  • the primary antibody used for the immunoblots was made against full length OP-1 produced in E.coli using standard recombinant DNA and antibody production techniques (see, for example, Example 8, below for a general description for producing morphogen-specific antibodies.) Fractions found to contain the morphogen then were applied to the next column step or used in the immunoreactivity or activity assays described below.
  • the S-Sepharose purification step was performed as follows. 200 ml of cation exchanger (S-Sepharose, Sigma Chemical Corp.) were equilibrated with equilibration buffer (6M urea, 20 mM MES, 70 mM NaCl, pH 6.5). The supernatant from the centrifuged extract was diluted to final concentration of 6M urea, 20 mM MES, 70 mM NaCl, pH 6.5. After loading, the column was washed to baseline using equilibration buffer, and the bound components were eluted stepwise from the column with 6M urea, 20 mM MES, 100 mM and 500 mM NaCl, pH 6.5. The more tightly bound components then were eluted with 4M guanidine, 20 mM sodium phosphate, pH 7.0.
  • equilibration buffer 6M urea, 20 mM MES, 70 mM NaCl, pH 6.5.
  • the Phenyl-Sepharose purification step was performed as follows. 15 ml of Phenyl-Sepharose CL-4B (Sigma) were equilibrated with 6M urea, 20 mM HEPES, 1M ammonium sulfate, 300 mM NaCl, pH 7.0. The 500 mM NaCl eluate from the S-Sepharose step was diluted with 6M urea, 20 mM HEPES, 3M ammonium sulfate, 300 mM NaCl, pH 7.0, to a final concentration of 1M ammonium sulfate, pH 7.0. After loading, the column was washed to baseline with equilibration buffer.
  • the column was eluted with 6M urea, 20 mM HEPES, 0.6M ammonium sulfate, 300 mM NaCl, pH 7.0, and then with 4M guanidine, 20 mM sodium phosphate, pH 7.0.
  • Scheme B The alternative S-Sepharose purification was performed as follows. Ethanol-precipitated protein was loaded onto an S-Sepharose column equilibrated in 6M urea, 20 mM MES, 50 mM NaCl, pH 6.5. Elution was stepwise with 6M urea, 20 mM MES, 100 mM NaCl and then 500 mM NaCl, and finally 4M guanidine, 20 mM sodium phosphate, pH 7.0. The Phenyl-Sepharose column was run as described above, with the 0.0M ammonium sulfate eluate then applied to a Cu++-IMAC column.
  • the Cu++IMAC purification step was performed as follows. 10 ml of Pharmacia Fast Flow Chelating Resin were charged with 0.2M cupric sulfate, and equilibrated with 6M urea, 20 mM HEPES, 0.5M NaCl, pH 7.0. After loading, the column was washed to baseline with equilibration buffer. Elution from the column was stepwise, using equilibration buffer containing 1 mM, 5 mM, or 10 mM imidazole. The column then was stripped with equilibration buffer containing 10 mM EDTA. The 10 mM imidazole elution was dialyzed against water/0.1% TFA, then against 30% acetonitirile/0.1% TFA.
  • the C-18 reverse phase chromatography purification step was performed as follows. An HPLC C-18 semi-prep column was used for the final purification step. The gradient used was 30-50% acetonitrile/0.1% TFA over 60 minutes at 3 ml/minutes. After the sample was loaded, the column was washed to baseline with 30% acetonitrile/0.1% TFA before the gradient is started. Fractions collected were 3 ml in size. Chromatograms were read at 214 nm.
  • Mammary glands were obtained from 2 female Long Evan rats (Charles River Labs, Wilmington, Mass.) one week post-partum. The excised glands were mildly homogenized in 6M urea, 20 mM methylethansulfonate (MES), 0.5M NaCl, pH 6.5 using a polytron homogenizer. The suspension then was centrifuged for 20 minutes at 8,000 RPM, and the supernatant removed for further purification.
  • MES mM methylethansulfonate
  • FIG. 1(A) shows the chromatogram and 1(B) the corresponding Western blot for fractions from the C-18 reverse phase chromatography step run under reducing conditions.
  • Lane S of the Western blot is a standard, containing reduced, purified, recombinantly-produced OP-1.
  • the arrows show molecular weight markers corresponding to 17, 27, and 39 Kd.
  • the reduced monomer run at approximately 16-18 Kd; the oxidized homodimer at approximately 36 Kd.
  • Lanes 13-30 represent the corresponding fractions of the C-18 reverse phase column as numbered in FIG. 1(A).
  • mammary extract OP-1 elutes primarily in fractions 21-25 from this final chromatography step.
  • Colostrum is the first milk to be produced by the mother following birth. Approximately 5 gallons of bovine colostrum were obtained from a local dairy farm and delipidated by centrifugation (8000 rpm for approximately 10 min. at 4° C.). The supernatant then was filtered through cheese cloth. The filtered supernatant was stored in 500 ml aliquots at 70° C.
  • 50 ml of colostrum were diluted with 100 ml of 9M urea, 30 mM sodium phosphate, pH 7.0. Alternately, 50 ml of colostrum was added to 50 ml of 8M guanidine-HCl, 50 mM Tris, pH 7.2 and precipitated with 40%, then 85% ice cold ethanol. The pellet was washed with 90% cold ethanol and lyophilized overnight. The lyophilized pellet was resuspended in 6M urea, 20 mM MES, 500 mM NaCl, pH 6.5, stirred overnight at 4° C., and centrifuged at 9,000 RPM for 10 minutes to clarify the suspension before loading onto the column as described in schemes A and B, above.
  • FIG. 2 shows the chromatogram (A) and corresponding Western blot (B) for results of purification scheme A (FIG. 2B-1, reduced and FIG. 2B-2, oxidized); and FIG. 3 shows the chromatogram (A) and Western blot (B, reduced) for C-18-purified protein from scheme B.
  • lane S in FIGS. 2B and 3B is a standard, containing purified, recombinantly produced OP-1; 17, 27 and 39 are molecular weight markers, and lane numbers correspond to fraction numbers in the corresponding chromatograms.
  • OP-1 purified by scheme A appears predominantly in fractions 18-27
  • OP-1 purified by scheme B appears predominantly in fractions 18-25.
  • Milk was obtained from the same cow from which the colostrum came, 57 days after the birth of the calf.
  • the milk was delipidated by centrifugation at 10,000 RPM for 15 minutes, and the milk was poured off and away from the fat layer.
  • the Phenyl-Sepharose column then was run as described above.
  • the Phenyl-Sepharose-bound sample was eluted and applied to a Cu++IMAC column, prepared and run as described above.
  • the 10 mM imidazole eluate was found to contain OP-1 and was dialyzed for further purification on the C-18 column.
  • lane S is a standard, containing purified, recombinantly produced OP-1; 17, 27, and 39 are molecular weight markers, and the lane numbers correspond to the fractions numbers in FIG. 4A.
  • OP-1 purified from 57-day milk appears predominantly in fractions 18-26.
  • OP-1 purified from the different milk sources as described above also were characterized by Western blotting using antibodies raised against OP-1 and BMP2.
  • Antibodies were prepared using standard immunology protocols well known in the art, and as described in Example 8, below using full-length E. coli-produced OP-1 and BMP2 as the immunogens.
  • OP-1 purified from colostrum reacts with the anti-OP-1 antibody, but not with anti-BMP2 antibody.
  • lane 1 contains reduced, purified, recombinantly-produced OP-1;
  • lane 2 contains C-18 purified bovine colostrum, and
  • lane 3 contains reduced COP-16, a biosynthetic construct having morphogenic activity and an amino acid sequence modeled on the proteins described herein, but having highest amino acid sequence homology with BMP2 (see U.S. Pat. No. 5,011,691 for the COP-16 amino acid sequence.)
  • FIG. 5A the gel was probed with anti-OP-1 antibody; in FIG. 5B, the gel was probed with anti-BMP2 antibody.
  • anti-OP-1 antibody hybridizes with protein in lanes 1 and 2, but not 3; while anti-BMP2 antibody hybridizes with lane 3 only.
  • C-18 purified mammary gland extract and 57-day milk also were shown to react with anti-OP-1 antibodies, including antibody raised against the full length E. coli OP-1, full length mammalian-produced OP-1, and the OP-1 Ser-17-Cys peptide (e.g., the OP-1 N-terminal 17 amino acids).
  • the morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo as follows. 33% of each OP-1 immunoreactive fraction of C-18-purified mammary gland extract was lyophilized and resuspended in 220 ⁇ l of 50% acetonitrile/0.1% TFA. After vortexing, 25 mg of collagen matrix was added. The samples were lyophilized overnight, and implanted in Long Evans rats (Charles River Laboratories, Wilmington, Mass., 28-35 days old). Each fraction was implanted in duplicate. For details of the collagen matrix implantation procedure, see, for example, U.S. Pat. No. 4,968,590, hereby incorporated by reference. After 12 days, the implants were removed and evaluated for new bone formation by histological observation.
  • FIG. 6A The results are presented in FIG. 6A, where "% activity" refers to the % of bone formation/total area covered by bone in the histology sample.
  • solid bars represent implants using mammary extract-derived OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see FIG. 1B), and the hatched bar represents implants using recombinantly produced OP-1 (600 ng).
  • the results demonstrate that the peak bone forming activity of C-18-purified mammary gland extract corresponds with the immunoreactive fraction peaks of FIG. 1B (compare FIG. 6A and 1B.)
  • OP-1 purified from mammary gland extract was evaluated in vitro by measuring alkaline phosphatase activity in vitro using the following assay.
  • Test samples were prepared using 15-20% of individual immunoreactive fractions from the C-18 run which were precipitated and resuspended in a smaller volume of 50% acetonitrile/0.1% TFA.
  • Alkaline phosphatase activity was tested using ROS 17/2.8 cells (Rat Osteosarcoma, e.g., obtained, for example, from Dr. Robert J. Majeska, Mt. Sinai Medical Center, New York, N.Y., in a standard alkaline phosphatase activity assay (see, for example, U.S. Pat. No.
  • FIG. 6B solid bars represent assays performed with mammary gland-purified OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see FIG. 1B), the hatched bar represents the assay performed with purified, recombinantly-produced OP-1 (100 ng/ml), and the cross-hatched bar represents background.
  • alkaline phosphatase activity corresponds with immunoreactivity of the C-18-purified extract (compare FIG. 6B and 1B.)
  • OP-1 was detected in human serum using the following assay.
  • activated agarose gel e.g., Affi-GelTM, from Bio-Rad Laboratories, Richmond, Calif., prepared following manufacturer's instructions
  • FIG. 7 is an immunoblot showing OP-1 in human sera under reducing and oxidized conditions.
  • lanes 1 and 4 are OP-1 standards, run under oxidized (lane 1) and reduced (lane 4) conditions.
  • Lane 5 shows molecular weight markers at 17, 27 and 39 kDa.
  • Lanes 2 and 3 are human sera OP-1, run under oxidized (lane 2) and reduced (lane 3) conditions.
  • CAMs are morphoregulatory molecules identified in all tissues as an essential step in tissue development.
  • N-CAMs which comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where "180", “140” and “120” indicate the apparent molecular weights of the isoforms as measured by polyacrylamide gel electrophoresis) are expressed at least transiently in developing tissues, and permanently in nerve tissue. Both the N-CAM-180 and N-CAM-140 isoforms are expressed in both developing and adult tissue. The N-CAM-120 isoform is found only in adult tissue. Another neural CAM is L1.
  • CAMs are implicated in normal tissue development; N-CAMs are implicated in appropriate neural development, including appropriate neurulation, neuronal migration, fasciculation, and synaptogenesis. Inhibition of N-CAM production, as by complexing the molecule with an N-CAM-specific antibody, inhibits retina organization, including retinal axon migration, and axon regeneration in the peripheral nervous system, as well as axon synapses with target muscle cells.
  • CAMs also have been postulated as part of a morphoregulatory pathway whose activity is induced by a to date unidentified molecule (See, for example, Edelman, G. M. (1986) Ann. Rev. Cell Biol. 2: 81-116). Without being limited to any given theory, the morphogens described herein may act as the inducer of this pathway.
  • the morphogens described herein can stimulate CAM production. As described below, the morphogens stimulate L1 and N-CAM production, including all three isoforms of the N-CAM molecule, in nerve tissue. A detailed description of this protocol is described in copending U.S. Ser. No. 922,813 the disclosure of which is incorporated hereinabove by reference.
  • NG108-15 cells were cultured for 4 days in the presence of increasing concentrations of OP-1 and standard Western blots performed on whole cells extracts.
  • the NG108-15 cell line is a hybrid cell line (neuroblastoma x glioma, American Type Culture Collection, Rockville, Md.).
  • N-CAM isoforms were detected with an antibody which crossreacts with all three isoforms, mAb H28.123, obtained from Sigma Chemical Co., St. Louis, the different isoforms being distinguishable by their different mobilities on an electrophoresis gel.
  • Control NG108-15 cells express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined by western blot analyses using up to 100 ⁇ g of protein.
  • treatment of NG108-15 cells with OP-1 resulted in a dose-dependent increase in the expression of the 180 kDa and 140 kDa isoforms, as well as the induction of the 120 kDa isoform.
  • FIG. 8B is a Western blot of OP1-treated NG108-15 cell extracts, probed with mAb H28.123, showing the induction of all three isoforms.
  • N-CAM-180 and N-CAM-140 induction are dose response curves of N-CAM-180 and N-CAM-140 induction as a function of morphogen concentration.
  • N-CAM-120 is not shown in the graph as it could not be quantitated in control cells.
  • N-CAM-120 is induced in response to morphogen treatment.
  • the induction of the 120 isoform also indicates that morphogen-induced redifferentiation of transformed cells stimulates not only redifferentiation of these cells from a transformed phenotype, but also differentiation to a phenotype corresponding to a developed cell.
  • the differential induction of N-CAM 180 and 140 isoforms seen may be because constitutive expression of the 140 isoform is close to maximum.
  • the increase in N-CAM expression corresponded in a dose-dependent manner with the morphogen induction of multicellular aggregates.
  • the cell aggregation effects of OP-1 on NG108-15 cells can be inhibited with anti-N-CAM antibodies or antisense N-CAM oligonucleotides.
  • Antisense oligonucleotides can be made synthetically on a nucleotide synthesizer, using standard means known in the art.
  • phosphorothioate oligonucleotides are prepared, to enhance transport of the nucleotides across cell membranes. Concentrations of both N-CAM antibodies and N-CAM antisense oligonucleotides sufficient to inhibit N-CAM induction also inhibited formation of multilayered cell aggregates.
  • soluble morphogen e.g., mature OP-1 associated with its pro domain
  • soluble morphogen e.g., mature OP-1 associated with its pro domain
  • At least one morphogen is found principally in early developing embryos (8-day embryos). As described below, morphogen neutralization with morphogen-specific antibodies inhibits embryogenesis.
  • Morphogen inhibition in developing embryos inhibits tissue and organ development. Specifically, 9-day mouse embryo cells, cultured in vitro under standard culturing conditions, were incubated in the presence and absence of an OP-1-specific monoclonal antibody prepared using recombinantly produced, purified mature OP-1 as the immunogen. The antibody was prepared using standard antibody production means well known in the art and essentially as described for Example 9, below. After two days, the effect of the antibody on the developing embryo was evaluated by histology using standard histology procedures well known in the art. As determined by histological examination, the OP-1-specific antibody specifically inhibits eye lobe formation in the developing embryo. In particular, the diencephalon outgrowth does not develop. In addition, the heart is malformed. Moreover, in separate immunolocalization studies on embryo sections with labelled OP-1 specific antibody, the OP-1-specific antibody localizes to neural epithelia.
  • morphogen activity may be demonstrated in fetal development in the mouse model using the following assay.
  • Single lip injections comprising 10-100 ⁇ g/injection of morphogen-specific antibody are administered to pregnant female mice during each day of the gestation period and tissue development (e.g., bone development) in treated and control new mice evaluated by standard histomorphometric analysis at birth.
  • anti-morphogen monoclonal and/or polyclonal antibodies may be prepared using standard methodologies including, for example, the protocol provided in Example 8, below, and provided to juveniles to inhibit the activity of endogenous morphogens.
  • mice e.g., 10-100 ⁇ g/injection/day for 10-15 days.
  • the mice are sacrificed and the effect of morphogen on bone development assessed by body weight, gross visual examination and histology.
  • anti-OP-1 antibodies were used in 10 ⁇ g injections/day for 14 days, and the mice were sacrificed at 21 days.
  • mice treated with OP-1 specific antibody show consistent and significant stunted growth, including reduced body length and body weight, (9B) as compared with untreated mice (9A). Histological examination showed reduced bone growth as evidenced by reduced bone size in the treated mice.
  • single lip injections also may be provided to older juveniles and adult mice (e.g., 10-100 ⁇ g) over a prolonged time (e.g., 10-15 days) to evaluate the effect or morphogen neutralization on bone growth and bone integrity and to evaluate the onset of osteoporosis.
  • ovariectomized rats Forty Long-Evans rats (Charles River Laboratories, Wilmington) weighing about 200 g each are ovariectomized (OVX) using standard surgical procedures, and ten rats are sham-operated. The ovariectomization of the rats produces an osteoporotic condition within the rats as a result of decreased estrogen production. Food and water are provided ad libitum. Eight days after ovariectomy, the rats, prepared as described above, were divided into five groups: (A), 10 sham-operated rats; (B), 10 ovariectomized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v.
  • PBS phosphate-buffered saline
  • OP-1 was the morphogen tested.
  • each rat was injected with 5 mg of tetracycline, and on day 22, the rats were sacrificed.
  • the body weights, uterine weights, serum alkaline phosphate levels, serum calcium levels and serum osteocalcin levels then were determined for each rat. The results are shown in Tables III and IV.
  • the rats were divided into four groups: (A) sham-operated (1 ml of PBS by intravenous injection through tail vein (5 rats); (B) OVX, into which nothing was injected (5 rats); (C) OVX, receiving about 1 mg of 17 ⁇ E 2 by intravenous injection through the tail vein (5 rats), and (D) OVX, receiving about 1 ⁇ g of morphogen by intravenous injection through the tail vein (5 rats).
  • OP-1 was morphogen tested.
  • the rats were injected daily as described for seven days, except no injections were given on the thirteenth day. The rats then were sacrificed on the nineteenth day. The tibial diaphyseal long bones then were removed and fixed in ethanol and histomorphometric analysis was carried out using standard procedures well known in the art. The results are shown in Table V.
  • Determining the tissue distribution of morphogens may be used to identify different morphogens expressed in a given tissue, as well as to identify new, related morphogens. Tissue distribution also may be used to identify useful morphogen-producing tissue for use in screening and identifying candidate morphogen-stimulating agents.
  • the morphogens (or their mRNA transcripts) readily are identified in different tissues using standard methodologies and minor modifications thereof in tissues where expression may be low. For example, protein distribution may be determined using standard Western blot analysis or immunofluorescent techniques, and antibodies specific to the morphogen or morphogens of interest. Similarly, the distribution of morphogen transcripts may be determined using standard Northern hybridization protocols and transcript-specific probes.
  • any probe capable of hybridizing specifically to a transcript, and distinguishing the transcript of interest from other, related transcripts may be used. Because the morphogens described herein share such high sequence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe specific for the pro region of the immature protein and/or the N-terminal region of the mature protein. Another useful sequence is the 3' non-coding region flanking and immediately following the stop codon. These portions of the sequence vary substantially among the morphogens of this invention, and accordingly, are specific for each protein.
  • Vgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bp fragment encoding both a portion of the untranslated pro region and the N-terminus of the mature sequence (see Lyons et al. (1989) PNAS 86: 4554-4558 for a description of the cDNA sequence).
  • mOP-1-specific probe sequences are the BstX1l-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb sequence immediately upstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an 0.3 Kb fragment containing a portion of the 3'untranslated sequence (See Seq. ID No. 18, where the pro region is defined essentially by residues 30-291.) Similar approaches may be used, for example, with hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and 22.)
  • morphogen transcripts can be identified in mammalian tissue, using standard methodologies well known to those having ordinary skill in the art. Briefly, total RNA is prepared from various adult murine tissues (e.g., liver, kidney, testis, heart, brain, thymus and stomach) by a standard methodology such as by the method of Chomczyaski et al. ((1987) Anal. Biochem 162: 156-159) and described below. Poly (A)+RNA is prepared by using oligo (dT)-cellulose chromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).
  • oligo (dT)-cellulose chromatography e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.
  • Poly (A)+RNA (generally 15 ⁇ g) from each tissue is fractionated on a 1% agarose/formaldehyde gel and transferred onto a Nytran membrane (Schleicher & Schuell). Following the transfer, the membrane is baked at 80° C. and the RNA is cross-linked under UV light (generally 30 seconds at 1 mW/cm 2 ). Prior to hybridization, the appropriate probe is denatured by heating. The hybridization is carried out in a lucite cylinder rotating in a roller bottle apparatus at approximately 1 rev/min for approximately 15 hours at 37° C. using a hybridization mix of 40% formamide, 5 ⁇ Denhardts, 5 ⁇ SSPE, and 0.1% SDS. Following hybridization, the non-specific counts are washed off the filters in 0.1 ⁇ SSPE, 0.1% SDS at 50° C.
  • kidney-related tissue appears to be the primary expression source for OP-1, with brain, heart and lung tissues being secondary sources.
  • Lung tissue appears to be the primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3.
  • Lower levels of Vgr-1 also are seen in kidney and heart tissue, while the liver appears to be a secondary expression source for BMP5, and the spleen appears to be a secondary expression source for BMP4.
  • GDF-1 appears to be expressed primarily in brain tissue.
  • OP-1 also is detected in adult rat stomach and gut tissue.
  • OP-2 appears to be expressed primarily in early embryonic tissue. Specifically, northern blots of murine embryos and 6-day post-natal animals shows abundant OP2 expression in 8-day embryos. Expression is reduced significantly in 17-day embryos and is not detected in post-natal animals.
  • labelled soluble OP-1 (iodinated with 125 I, using standard labelling procedures well known in the art) and injected into the rat tail vein also is localized to the stomach tissue within 30 minutes of injection.
  • Morphogens are readily detected in solution with a standard immunoassay, using a polyclonal or monoclonal antibody specific for that protein and standard Western blot, ELISA (enzyme-linked immunoabsorbant assay) or other immunoassay technique well known in the art.
  • ELISA enzyme-linked immunoabsorbant assay
  • a currently preferred, exemplary protocol for an ELISA assay, as well as means for generating morphogen-specific antibody are presented below. Standard protocols for antibody production, Western blot and other immunoassays also are described, for example, in Molecular Cloning A Laboratory Manual, Sambrook et al., eds. 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Standard ELISA technique is described, for example, by Engvall (1980) Methods Enzymol. 70: 419-439.
  • Polyclonal antibody was prepared as follows. Each rabbit was given a primary immunization of 100 ⁇ g/500 ⁇ l E. coli-produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 ⁇ l Complete Freund's Adjuvant. The antigen was injected subcutaneously at multiple sites on the back and flanks of the animal. The rabbit was boosted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional boosts and test bleeds were performed at monthly intervals until antibody against OP-1 was detected in the serum using an ELISA assay. Then, the rabbit was boosted monthly with 100 ⁇ g of antigen and bled (15 ml per bleed) at days seven and ten after boosting.
  • Monoclonal antibody specific for a given morphogen was prepared as follows. A mouse was given two injections of E. coli produced OP-1 monomer. The first injection contains 100 ⁇ g of OP-1 in complete Freund's adjuvant and was given subcutaneously. The second injection contained 50 ⁇ g of OP-1 in incomplete adjuvant and was given intraperitoneally. The mouse then received a total of 230 ⁇ g of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period.
  • mice were boosted intraperitoneally with 100 ⁇ g of OP-1 (307-431) and 30 ⁇ g of the N-terminal peptide (Ser293-Asn309-Cys) conjugated through the added cysteine to bovine serum albumin with SMCC crosslinking agent. This boost was repeated five days (IP), four days (IP), three days (IP) and one day (IV) prior to fusion.
  • the mouse spleen cells then were fused to commercially available myeloma cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the cell fusion plated and screened for OP-1-specific antibodies using OP-1 (307-431) as antigen.
  • the cell fusion and monoclonal screening then were according to standard procedures well described in standard texts widely available in the art e.g., Maniatis et al. Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press.

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Abstract

Disclosed are methods and compositions useful in dietary applications and capable of enhancing tissue morphogenesis, including tissue development and viability in a mammal, particularly a human. The methods and compositions include a morphogen which, when provided to an individual as a food formulation or supplement, is capable of enhancing tissue development and viability in the individual.

Description

CROSS REFERENCE TO RELAYED APPLICATIONS
This application is a continuation of Ser. No. 07/946,235, filed Sep. 16, 1992, now abandoned, which is continuation-in-part of (1) U.S. Ser. No. 07/922,813, filed Jul. 31, 1992, now abandoned which is a continuation-in-part of U.S. Ser. No. 07/752,764, filed Aug. 30, 1991, now abandoned, which in turn is a continuation in part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991, now abandoned; (2) U.S. Ser. No. 07/923,780, filed Jul. 31, 1992, now abandoned which is a continuation-in-part of U.S. Ser. No. 07/752,857, filed Aug. 30, 1991, now abandoned which in turn is a continuation-in-part of U.S. Ser. No. 07/667,274 filed Mar. 11, 1991, now abandoned; (3) U.S. Ser. No. 07/938,336 and U.S. Ser. No. 07/938,337, both, filed Aug. 28, 1992, both now abandoned and both continuations-in-part of U.S. Ser. No. 07/753,059, filed Aug. 30, 1991, now abandoned which in turn is a continuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991, now abandoned; and (4) U.S. Ser. No. 07/752,764, filed Aug. 30, 1991, now abandoned which is a continuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991, now abandoned. The disclosures of these applications are incorporated herein by reference. The disclosure of U.S. Ser. No. 07/946,238, filed Sep. 16, 1992, now abandoned, and currently pending as U.S. Ser. No. 08/445,468, filed May 22, 1995, now U.S. Pat. No. 5,849,686, is also incorporated herein by reference.
FIELD OF THE INVENTION
This invention relates generally to the field of dietary compositions and supplements.
BACKGROUND OF THE INVENTION
The present invention relates to compositions useful as mammalian dietary compositions and supplements. In particular, the invention relates to food additives and dietary supplements capable of enhancing tissue morphogenesis and development, particularly in individuals at risk for normal tissue development and viability. Examples of such individuals include infants, particularly prematurely-born ("preterm") and low birth weight infants, and juveniles; aged individuals; and individuals experiencing altered metabolic function and/or suffering from metabolic dysfunctions and other disorders that threaten organ or tissue function or viability, such as can result from malnutrition or starvation, autoimmune diseases, organ cirrhosis and other tissue necrotizing dysfunctions, or disorders associated with aging cells (cell senescence.)
Mammalian infants are nourished by mother's milk until such time as they can digest food solids. Infant formulas now exist for humans and other mammals which can supplant or supplement mother's milk. The formulas may be milk based (e.g., cow milk) or non-milk-based (e.g., soy). Particularly at risk are prematurely born infants whose tissues and organs are at an earlier stage of development, and whose nutritional requirements may differ from those of full term infants. Formula development is an ongoing endeavor to more accurately mimic the beneficial aspects of mother's milk. Nevertheless, despite the efforts of many researchers, infant formulas still differ in a number of significant ways from human milk. In part this is due because human milk has many substances, such as immunoglobulins, free amino acids, polyamines, nucleotides and polyunsaturated fatty acids not present, for example, in cow's milk. In addition, while infant formulas try to mimic the protein quantity found in human milk, the foreign proteins typically are present in the formula as hydrolysates to avoid rejection or reaction by the infant's digestive system. The proteins are present primarily as amino acid sources rather than as functional proteins as might normally be transmitted by the nursing mother to the infant. In addition, human milk may contain unidentified growth and differentiation factors that are important for overall tissue and skeletal development.
Another group of individuals with potentially unique nutritional requirements are individuals undergoing metabolic changes which may result from periods of intense growth or stress, including, for example, pregnant women and drowning victims. Other sources of stress to the body may result from malnutrition or starvation, or from metabolic disorders that affect organ viability, such as autoimmune disease and organ cirrhosis. Aged individuals, and postmenopausal women also have altered or slower metabolic function. All of these individuals are at risk for tissue damage or loss of tissue function due to altered metabolic function.
Reduced or lost tissue function due to malnutrition also is found in many patients admitted to hospitals (protein energy malnutrition, "PEM"). Proper nutritional support for such patients, while not a primary mode of treatment is, nevertheless, an important factor for therapy and recovery. It is, therefore important to administer a nutritionally balanced diet given orally, enterally or parenterally, adequate to the needs of the patient. This is especially true for those patients where conventional feeding is contraindicated (e.g., in dehydrated or gastroenterological patients) or is insufficient (e.g., in hypercatabolic patients). The enteral or oral mode of administration of foods typically is preferable to parenteral modes because of the lower morbidity, trophic effect upon the intestinal mucosa, reduced dependency on instrumentation and lower costs.
It is an object of this invention to provide dietary compositions and supplements for enhancing tissue morphogenesis, including tissue growth, development, maintenance and viability in a mammal, particularly a human. Another object of the invention is to provide an infant formula capable of enhancing tissue development in an infant or juvenile. Still another object is to provide an infant formula that more closely mimics a nursing mother's milk. Another object of the invention is to provide dietary supplements for individuals at risk for normal tissue development, growth, maintenance and viability, including premature infants, aged individuals and individuals with altered metabolic function and/or suffering from disorders or metabolic dysfunctions which threaten organ viability and function. These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods useful in dietary applications and capable of enhancing tissue morphogenesis, including tissue growth, development, maintenance and viability in a mammal, particularly a human. The dietary compositions and supplements of this invention comprise a morphogenic protein ("morphogen"), as described herein, which, when provided to an individual as a food formulation or supplement, is capable of enhancing tissue development, growth, maintenance and/or viability in the individual. The compositions and processes provided herein are suitable for both infants and adults, and as part of clinical nutrition.
As used herein, "enhancing tissue viability" is understood to mean protecting tissue from lost or reduced tissue function due to cell damage or cell senescence, including inducing cells to maintain their differentiated phenotype, inducing regeneration of damaged tissue, and/or inhibiting additional damage thereto. "Morphogenically effective concentration" is understood to mean a concentration sufficient to enhance tissue development and tissue viability in an individual at risk for tissue damage and/or reduced or lost tissue function due to insufficient nutritional considerations, tissue damage associated therewith, and/or incomplete tissue development, regardless of etiology. The ability of morphogens to repair, regenerate and protect various disparate tissues, including but not limited to, tissues of the gastrointestinal tract, including the oral mucosa, liver tissue, dentin tissue, periodontal tissue, nerve tissue, bone tissue, and any tissue at risk of damage due to immune response-mediated tissue destruction, including ischemia-reperfusion related tissue damage are disposed in copending U.S. Ser. No. 07/945,286 filed Sep. 15, 1992, (currently pending as U.S. Ser. No. 08/461,397), U.S. Ser. No. 07/946,238, filed Sep. 16, 1992 (currently pending as U.S. Ser. No. 08/445,468), U.S. Ser. No. 07/752,764, filed Aug. 30, 1991 (currently pending as U.S. Ser. No. 08/404,113), U.S. Ser. No. 07/945,285, filed Sep. 15, 1992 (currently pending as U.S. Ser. No. 08/155,343), U.S. Ser. No. 07/922,813, filed Jul. 31, 1992 (currently pending as U.S. Ser. No. 08/260,675), U.S. Ser. No. 07/923,780, filed Jul. 31, 1992 (currently pending as U.S. Ser. No. 08/432,883), and U.S. Ser. No. 07/938,336, filed Aug. 2, 1992 (currently pending as U.S. Ser. No. 08/445,467), respectively, the disclosures of which are incorporated herein above by reference. "Morphogen-solubilizing molecule" is understood to mean a molecule capable of maintaining a morphogen in soluble form in physiologically buffered solutions. "Food formulation" is understood to mean a dietary composition normally ingested by an individual to satisfy the body's fundamental nutritional requirements; "dietary supplement" is understood to mean supplemental compositions ingested by an individual in addition to the food formulations ingested to satisfy the fundamental nutritional requirements. Multivitamin and iron tablets are examples of common dietary supplements. "Dietary composition" is understood to include both food formulations and dietary supplements. As used herein, the term "infant formula" is understood to refer to the well established infant compositions as defined by the American Academy of Pediatrics (AAP) and the AAP Committee on Nutrition ((1985) Pediatrics 75: 976, the European Society of Pediatric Gastroenterology and Nutrition (ESPGAN) and the ESPGAN Committee on Nutrition ((1987) Acta Paed Scan Suppl: 330), including recent updates published by these committees on infant formula nutritional guidelines.
The dietary composition or supplement preferably is administered orally, and may be provided in liquid form or as a powder to be dissolved in a beverage. Alternatively, the dietary supplement may be provided as a solid, e.g., in a capsular, tablet, troche or lozenge form; or, the supplement may be provided as an aerosol, for oral or nasal administration. Where oral administration is not possible or desirable, other administration routes are envisioned. For example, for some premature infants, or for intubated patients, parenteral administration may be required, e.g., via an enteral feeding tube.
The morphogen may be provided alone or in association with one or more suitable excipients or carriers, and/or in combination with other beneficial molecules such as vitamins, minerals, lipids, fiber sources and the like. The dietary supplements also may include pharmaceutically acceptable inert materials for use as binders or stabilizers, including magnesium stearate or calcium carbonate. The morphogen may be formulated together with one or more normal food ingredients, e.g., as part of a food formulation. Alternatively or, in addition, the morphogen may be provided as a dietary supplement in, for example, tablet or syrup form.
The mature form of the morphogen, or active truncated forms thereof which may be formulated in the composition, further may be provided in association with a morphogen precursor "pro" domain, which is known to enhance the solubility of the protein in physiologically buffered solutions. Other useful molecules known to enhance protein solubility include casein, including derivatives, salts and analogs thereof, as well as other milk components, and various serum and milk serum proteins. Additional useful molecules which may be associated with the morphogen include tissue targeting molecules capable of directing the morphogen to a desired target tissue. Tissue targeting molecules envisioned to be useful in the treatment protocols of this invention include antibodies, antibody fragments or other binding proteins which interact specifically with surface molecules on the target tissue cells.
Still another useful tissue targeting molecule may be part or all of a morphogen precursor "pro" domain. Morphogens may be synthesized in one tissue and secreted and transported to another tissue. For example, while the protein has been shown to be active in bone tissue, the primary source of OP-1 synthesis appears to be the tissue of the urogenic system (e.g., renal and bladder tissue), with secondary expression levels occurring in the brain, heart, lungs and gastrointestinal tract (GI tract, see below.) Moreover, the protein has been identified in serum, saliva and various milk forms. In addition, the secreted form of the protein comprises the mature dimer in association with the pro domain of the intact morphogen sequence. Accordingly, the associated morphogen pro domains may act to target specific morphogens to different tissues in vivo. As described below, morphogen species comprising the pro domain may be obtained from the culture medium of morphogen-secreting mammalian cells. Alternatively, a tissue-targeting species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
Associated tissue targeting or solubility-enhancing molecules also may be covalently linked to the morphogen using standard chemical means.
In one preferred embodiment, the morphogen comprises part of an infant formula. The infant formula may be milk-based or nonmilk-based, e.g., soy-based. A typical ready-to-feed morphogen-enriched formulation for infants, when diluted to feeding concentrations, comprises, in addition to the morphogen added to the formula, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.
In another preferred embodiment, the dietary composition is formulated for individuals at risk for reduced or lost tissue function, such as postmenopausal women, elderly individuals, undernourished or malnourished individuals, dehydrated individuals, drowning victims, individuals suffering from metabolic disorders including an endocrine imbalance, gastrointestinal disorders, or immune-compromised individuals. Undernourished or malnourished individuals include those suffering from a lack of food (starvation) and/or eating disorders (e.g., anorexia nervosa), and/or suffering from a maladsorption syndrome (e.g., individuals afflicted with digestive or intestinal fistulas, shortened bowel, or hypercatabolism.) Individuals receiving a medical therapy, including radiotherapy, chemotherapy or a surgical procedure also are at risk for reduced or lost tissue function as a result of a therapy-related malabsorption-malnutrition dysfunction. In another embodiment, the dietary supplement is formulated for individuals undergoing periods of increased growth or stress, such as infants and juveniles, or pregnant or lactating women. In another embodiment, the dietary supplement is formulated for individuals at risk for reduced or lost organ function as results from tissue cirrhosis or an autoimmune disease.
Morphogen-enriched nutritional products, particularly clinical nutrition products for use in hospital or other clinical settings, in addition to comprising a morphogen preferably are based on the utilization of diverse other protein sources (casein, sodium and calcium caseinate, isolated soy protein, protein hydrolyzates and/or crystalline amino acids) mixtures of vegetable and animal fats, carbohydrates (basically glucose polymers), vitamins and minerals to meet, at least, the dietary intakes recommended for healthy individuals (see, for example, Committee on Dietary Allowances, Food and Nutrition Board, Nat Acad Sci, 9th Ed, 1980).
Among the morphogens useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, see Lee (1991) PNAS 88: 4250-4254), all of which are presented in Table II and Seq. ID Nos.5-14), and the recently identified 60A protein (from Drosophila, Seq. ID No. 24, see Wharton et al. (1991) PNAS 88: 9214-9218.) The members of this family, which include members of the TGF-β super-family of proteins, share substantial amino acid sequence homology in their C-terminal regions. The proteins are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature sequence. The "pro" form of the protein includes the pro domain and the mature domain, and forms a soluble species that appears to be the primary form secreted from cultured mammalian cells. The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14: 4683-4691.) Table I, below, describes the various morphogens identified to date, including their nomenclature as used herein, their Seq. ID references, and publication sources for the amino acid sequences for the full length proteins not included in the Seq. Listing. The disclosure of these publications is incorporated herein by reference.
              TABLE I                                                     
______________________________________                                    
"OP-1"     Refers generically to the group of                             
           morphogenically active proteins expressed                      
           from part or all of a DNA sequence                             
           encoding OP-1 protein, including allelic                       
           and species variants thereof, e.g., human                      
           OP-1 ("hOP-1", Seq. ID No. 5, mature                           
           protein amino acid sequence), or mouse                         
           OP-1 ("mOP-1", Seq. ID No. 6, mature                           
           protein amino acid sequence.) The                              
           conserved seven cysteine skeleton is                           
           defined by residues 38 to 139 of Seq. ID                       
           Nos. 5 and 6. The cDNA sequences and the                       
           amino acids encoding the full length                           
           proteins are provided in Seq. Id Nos. 16                       
           and 17 (hOP1 and Seq. ID Nos. 18 and 19                        
           (mOP1.) The mature proteins are defined                        
           by residues 293-431 (hOP1) and 292-430                         
           (mOP1). The "pro" regions of the                               
           proteins, cleaved to yield the mature,                         
           morphogenically active proteins are                            
           defined essentially by residues 30-292                         
           (hOP1) and residues 30-291 (mOP1).                             
"OP-2"     refers generically to the group of active                      
           proteins expressed from part or all of a                       
           DNA sequence encoding OP-2 protein,                            
           including allelic and species variants                         
           thereof, e.g., human OP-2 ("hOP-2", Seq.                       
           ID No. 7, mature protein amino acid                            
           sequence) or mouse OP-2 ("mOP-2", Seq. ID                      
           No. 8, mature protein amino acid                               
           sequence). The conserved seven cysteine                        
           skeleton is defined by residues 38 to 139                      
           of Seq. ID Nos. 7 and 8. The cDNA                              
           sequences and the amino acids encoding the                     
           full length proteins are provided in Seq.                      
           ID Nos. 20 and 21 (hOP2) and Seq. ID Nos.                      
           22 and 23 (mOP2.) The mature proteins are                      
           defined essentially by residues 264-402                        
           (hOP2) and 261-399 (mOP2). The "pro"                           
           regions of the proteins, cleaved to yield                      
           the mature, morphogenically active                             
           proteins likely are defined essentially by                     
           residues 18-263 (hOP2) and residues 18-260                     
           (mOP2). (Another cleavage site also                            
           occurs 21 residues upstream for both OP-2                      
           proteins.)                                                     
"CBMP2"    refers generically to the morphogenically                      
           active proteins expressed from a DNA                           
           sequence encoding the CBMP2 proteins,                          
           including allelic and species variants                         
           thereof, e.g., human CBMP2A ("CBMP2A(fx)",                     
           Seq ID No. 9) or human CBMP2B DNA                              
           ("CBMP2B(fx)", Seq. ID No. 10). The amino                      
           acid sequence for the full length                              
           proteins, referred to in the literature as                     
           BMP2A and BMP2B, or BMP2 and BMP4, appear                      
           in Wozney, et al. (1988) Science 242:1528-                     
           1534. The pro domain for BMP2 (BMP2A)                          
           likely includes residues 25-248 or 25-282;                     
           the mature protein, residues 249-396 or                        
           283-396. The pro domain for BMP4 (BMP2B)                       
           likely includes residues 25-256 or 25-292;                     
           the mature protein, residues 257-408 or                        
           293-408.                                                       
"DPP(fx)"  refers to protein sequences encoded by the                     
           Drosophila DPP gene and defining the                           
           conserved seven cysteine skeleton (Seq. ID                     
           No. 11). The amino acid sequence for the                       
           full length protein appears in Padgett, et                     
           al (1987) Nature 325: 81-84. The pro                           
           domain likely extends from the signal                          
           peptide cleavage site to residue 456; the                      
           mature protein likely is defined by                            
           residues 457-588.                                              
"Vgl(fx)"  refers to protein sequences encoded by the                     
           Xenopus Vgl gene and defining the                              
           conserved seven cysteine skeleton (Seq. ID                     
           No. 12). The amino acid sequence for the                       
           full length protein appears in                                 
           Weeks (1987) Cell 51: 861-867. The                             
           prodomain likely extends from the signal                       
           peptide cleavage site to residue 246; the                      
           mature protein likely is defined by                            
           residues 247-360.                                              
"Vgr-1(fx)"                                                               
           refers to protein sequences encoded by the                     
           murine Vgr-1 gene and defining the                             
           conserved seven cysteine skeleton (Seq. ID                     
           No. 13). The amino acid sequence for the                       
           full length protein appears in Lyons, et                       
           al, (1989) PNAS 86: 4554-4558. The                             
           prodomain likely extends from the signal                       
           peptide cleavage site to residue 299; the                      
           mature protein likely is defined by                            
           residues 300-438.                                              
"GDF-1(fx)"                                                               
           refers to protein sequences encoded by the                     
           human GDF-1 gene and defining the                              
           conserved seven cysteine skeleton (Seq. ID                     
           No. 14). The cDNA and encoded amino                            
           sequence for the full length protein is                        
           provided in Seq. ID. No. 32. The                               
           prodomain likely extends from the signal                       
           peptide cleavage site to residue 214; the                      
           mature protein likely is defined by                            
           residues 215-372.                                              
"60A"      refers generically to the morphogenically                      
           active proteins expressed from part or all                     
           of a DNA sequence (from the Drosophila 60A                     
           gene) encoding the 60A proteins (see Seq.                      
           ID No. 24 wherein the cDNA and encoded                         
           amino acid sequence for the full length                        
           protein is provided). "60A(fx)" refers to                      
           the protein sequences defining the                             
           conserved seven cysteine skeleton                              
           (residues 354 to 455 of Seq. ID No. 24.)                       
           The prodomain likely extends from the                          
           signal peptide cleavage site to residue                        
           324; the mature protein likely is defined                      
           by residues 325-455.                                           
"BMP3(fx)" refers to protein sequences encoded by the                     
           human BMP3 gene and defining the conserved.                    
           seven cysteine skeleton (Seq. ID No. 26).                      
           The amino acid sequence for the full                           
           length protein appears in Wozney et al.                        
           (1988) Science 242: 1528-1534. The pro                         
           domain likely extends from the signal                          
           peptide cleavage site to residue 290; the                      
           mature protein likely is defined by                            
           residues 291-472.                                              
"BMP5(fx)" refers to protein sequences encoded by the                     
           human BMP5 gene and defining the conserved                     
           seven cysteine skeleton (Seq. ID No. 27).                      
           The amino acid sequence for the full                           
           length protein appears in Celeste, et al.                      
           (1991) PNAS 87: 9843-9847. The pro domain                      
           likely extends from the signal peptide                         
           cleavage site to residue 316; the mature                       
           protein likely is defined by residues                          
           317-454.                                                       
"BMP6(fx)" refers to protein sequences encoded by the                     
           human BMP6 gene and defining the conserved                     
           seven cysteine skeleton (Seq. ID No. 28).                      
           The amino acid sequence for the full                           
           length protein appears in Celeste, et al.                      
           (1990) PNAS 87: 9843-5847. The pro domain                      
           likely includes extends from the signal                        
           peptide cleavage site to residue 374; the                      
           mature sequence likely includes                                
           residues 375-513.                                              
______________________________________
The OP-2 proteins have an additional cysteine residue in this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in addition to the conserved cysteine skeleton in common with the other proteins in this family. The GDF-1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq. ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. In addition, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton.
The morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention. Thus, as defined herein, a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5, including functionally equivalent arrangements of these cysteines (e.g., amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that, when the polypeptide chains are folded, the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein. Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells. In addition, it is also anticipated that these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.
In one preferred aspect, the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); where each Xaa indicates one of the 20 naturally-occurring L-isomer, α-amino acids or a derivative thereof. Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine identified in OP-2 (see residue 36, Seq. ID No. 2). In another preferred aspect, these sequences further comprise the following additional sequence at their N-terminus:
______________________________________                                    
Cys     Xaa     Xaa     Xaa   Xaa   (Seq. ID No. 15)                      
1                             5                                           
______________________________________
Preferred amino acid sequences within the foregoing generic sequences include: Generic Sequence 3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic Sequences accommodate the homologies shared among the various preferred members of this morphogen family identified in Table II, as well as the amino acid sequence variation among them. Specifically, Generic Sequences 3 and 4 are composite amino acid sequences of the following proteins presented in Table II and identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences include both the amino acid identity shared by the sequences in Table II, as well as alternative residues for the variable positions within the sequence. Note that these generic sequences allow for an additional cysteine at position 41 or 46 in Generic Sequences 3 or 4, respectively, providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids which influence the tertiary structure of the proteins.
 ______________________________________                                    
Generic Sequence 3                                                        
______________________________________                                    
      Leu    Tyr    Val  Xaa   Phe                                        
      1                        5                                          
Xaa   Xaa    Xaa    Gly  Trp   Xaa  Xaa  Trp  Xaa                         
                         10                                               
Xaa   Ala    Pro    Xaa  Gly   Xaa  Xaa  Ala                              
15                             20                                         
Xaa   Tyr    Cys    Xaa  Gly   Xaa  Cys  Xaa                              
             25                          30                               
Xaa   Pro    Xaa    Xaa  Xaa   Xaa  Xaa                                   
                         35                                               
Xaa   Xaa    Xaa    Asn  His   Ala  Xaa  Xaa                              
             40                          45                               
Xaa   Xaa    Leu    Xaa  Xaa   Xaa  Xaa  Xaa                              
                         50                                               
Xaa   Xaa    Xaa    Xaa  Xaa   Xaa  Xaa  Cys                              
      55                            60                                    
Cys   Xaa    Pro    Xaa  Xaa   Xaa  Xaa  Xaa                              
                    65                                                    
Xaa   Xaa    Xaa    Leu  Xaa   Xaa  Xaa                                   
70                             75                                         
Xaa   Xaa    Xaa    Xaa  Val   Xaa  Leu  Xaa                              
                    80                                                    
Xaa   Xaa    Xaa    Xaa  Met   Xaa  Val  Xaa                              
85                             90                                         
Xaa   Cys    Gly    Cys  Xaa                                              
             95                                                           
______________________________________
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: "Res." means "residue" and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50=(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala); Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gin or Glu); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr or Ala); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or Arg);
 ______________________________________                                    
Generic Sequence 4                                                        
______________________________________                                    
Cys   Xaa    Xaa    Xaa  Xaa  Leu  Tyr  Val  Xaa  Phe                     
1                        5                        10                      
Xaa   Xaa    Xaa    Gly  Trp  Xaa  Xaa  Trp  Xaa                          
                         15                                               
Xaa   Ala    Pro    Xaa  Gly  Xaa  Xaa  Ala                               
20                            25                                          
Xaa   Tyr    Cys    Xaa  Gly  Xaa  Cys  Xaa                               
             30                         35                                
Xaa   Pro    Xaa    Xaa  Xaa  Xaa  Xaa                                    
                         40                                               
Xaa   Xaa    Xaa    Asn  His  Ala  Xaa  Xaa                               
             45                         50                                
Xaa   Xaa    Leu    Xaa  Xaa  Xaa  Xaa  Xaa                               
                         55                                               
Xaa   Xaa    Xaa    Xaa  Xaa  Xaa  Xaa  Cys                               
      60                           65                                     
Cys   Xaa    Pro    Xaa  Xaa  Xaa  Xaa  Xaa                               
                    70                                                    
Xaa   Xaa    Xaa    Leu  Xaa  Xaa  Xaa                                    
75                            80                                          
Xaa   Xaa    Xaa    Xaa  Val  Xaa  Leu  Xaa                               
                    85                                                    
Xaa   Xaa    Xaa    Xaa  Met  Xaa  Val  Xaa                               
90                            95                                          
Xaa   Cys    Gly    Cys  Xaa                                              
             100                                                          
______________________________________
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: "Res." means "residue" and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly, Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln or Glu); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or Glu); and Xaa at res.102=(His or Arg).
Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among all the morphogen protein family members identified in Table II. Specifically, Generic Sequences 5 and 6 are composite amino acid sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq. ID Nos. 24-25). The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons ( Generic Sequences 5 and 6, respectively), as well as alternative residues for the variable positions within the sequence. As for Generic Sequences 3 and 4, Generic Sequences 5 and 6 allow for an additional cysteine at position 41 (Generic Sequence 5) or position 46 (Generic Sequence 6), providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.
 ______________________________________                                    
Generic Sequence 5                                                        
______________________________________                                    
      Leu    Xaa    Xaa  Xaa   Phe                                        
      1                        5                                          
Xaa   Xaa    Xaa    Gly  Trp   Xaa  Xaa  Trp  Xaa                         
                         10                                               
Xaa   Xaa    Pro    Xaa  Xaa   Xaa  Xaa  Ala                              
15                             20                                         
Xaa   Tyr    Cys    Xaa  Gly   Xaa  Cys  Xaa                              
             25                          30                               
Xaa   Pro    Xaa    Xaa  Xaa   Xaa  Xaa                                   
                         35                                               
Xaa   Xaa    Xaa    Asn  His   Ala  Xaa  Xaa                              
             40                          45                               
Xaa   Xaa    Xaa    Xaa  Xaa   Xaa  Xaa  Xaa                              
                         50                                               
Xaa   Xaa    Xaa    Xaa  Xaa   Xaa  Xaa  Cys                              
      55                            60                                    
Cys   Xaa    Pro    Xaa  Xaa   Xaa  Xaa  Xaa                              
                    65                                                    
Xaa   Xaa    Xaa    Leu  Xaa   Xaa  Xaa                                   
70                             75                                         
Xaa   Xaa    Xaa    Xaa  Val   Xaa  Leu  Xaa                              
                    80                                                    
Xaa   Xaa    Xaa    Xaa  Met   Xaa  Val  Xaa                              
85                             90                                         
Xaa   Cys    Xaa    Cys  Xaa                                              
             95                                                           
______________________________________
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: "Res." means "residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or Val); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa at res.97=(His or Arg).
 ______________________________________                                    
Generic Sequence 6                                                        
______________________________________                                    
Cys   Xaa    Xaa    Xaa  Xaa  Leu  Xaa  Xaa  Xaa  Phe                     
1                        5                        10                      
Xaa   Xaa    Xaa    Gly  Trp  Xaa  Xaa  Trp  Xaa                          
                         15                                               
Xaa   Xaa    Pro    Xaa  Xaa  Xaa  Xaa  Ala                               
20                            25                                          
Xaa   Tyr    Cys    Xaa  Gly  Xaa  Cys  Xaa                               
             30                         35                                
Xaa   Pro    Xaa    Xaa  Xaa  Xaa  Xaa                                    
                         40                                               
Xaa   Xaa    Xaa    Asn  His  Ala  Xaa  Xaa                               
             45                         50                                
Xaa   Xaa    Xaa    Xaa  Xaa  Xaa  Xaa  Xaa                               
                         55                                               
Xaa   Xaa    Xaa    Xaa  Xaa  Xaa  Xaa  Cys                               
      60                           65                                     
Cys   Xaa    Pro    Xaa  Xaa  Xaa  Xaa  Xaa                               
                    70                                                    
Xaa   Xaa    Xaa    Leu  Xaa  Xaa  Xaa                                    
75                            80                                          
Xaa   Xaa    Xaa    Xaa  Val  Xaa  Leu  Xaa                               
                    85                                                    
Xaa   Xaa    Xaa    Xaa  Met  Xaa  Val  Xaa                               
90                            95                                          
Xaa   Cys    Xaa    Cys  Xaa                                              
             100                                                          
______________________________________
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: "Res." means "residue" and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg, Asn or Glu); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at res.35=(Ala, Ser, Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp, Ala, Thr or Pro); Xaa at res.40=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala, Ser, Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.58=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.59=(Pro, Ser or Val); Xaa at res.60=(Glu, Asp, Gly, Val or Lys); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys, Leu or Glu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr, Ala or Glu); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala or Ile); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa at res.102=(His or Arg).
Particularly useful sequences for use as morphogens in this invention include the C-terminal domains, e.g., the C-terminal 96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as well as proteins comprising the C-terminal domains of 60A, BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at least the conserved six or seven cysteine skeleton. In addition, biosynthetic constructs designed from the generic sequences, such as COP-1, 3-5, 7, 16, disclosed in U.S. Pat. No. 5,011,691, also are useful. Other sequences include the inhibins/activin proteins (see, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other useful sequences are those sharing at least 70% amino acid sequence homology or "similarity", and preferably 80% homology or similarity with any of the sequences above. These are anticipated to include allelic and species variants and mutants, and biosynthetic muteins, as well as novel members of this morphogenic family of proteins. Particularly envisioned in the family of related proteins are those proteins exhibiting morphogenic activity and wherein the amino acid changes from the preferred sequences include conservative changes, e.g., those as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362, (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington, D.C. 1979). As used herein, potentially useful sequences are aligned with a known morphogen sequence using the method of Needleman et al. ((1970) J.Mol.Biol. 48: 443-453) and identities calculated by the Align program (DNAstar, Inc.). "Homology" or "similarity" as used herein includes allowed conservative changes as defined by Dayoff et al.
The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in another preferred aspect of the invention, useful morphogens include active proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as "OPX", which accommodates the homologies between the various identified species of OP1 and OP2 (Seq. ID No. 29).
The morphogens useful in the methods, composition and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and species variants of these proteins, naturally-occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
The morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells. A detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in copending U.S. patent application Ser. No. 752,764, filed Aug. 30, 1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of which are incorporated herein by reference.
Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins useful as dietary compositions for enhancing tissue morphogenesis, including enhancing tissue development and tissue viability in a variety of mammals, including humans.
The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects and features of this invention, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which:
FIG. 1A and B shows relative amounts of protein present in mammary gland extract eluate fractions of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot (B);
FIG. 2A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme A of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western blot under reduced (1) and oxidized (2) conditions (B);
FIG. 3A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme B of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot under reduced conditions (B);
FIG. 4A and B shows relative amounts of protein present in bovine 57 day milk eluate fractions of a C-18 reverse phase chromatography column (A), and the corresponding results of a Western Blot under reduced (1) and oxidized (2) conditions (B);
FIG. 5 shows Western Blot analysis of bovine colostrum using OP-1 and BMP2-specific antibodies;
FIG. 6A and B show results of in vivo and in vitro activity assays, respectively, for the corresponding fractions shown in FIG. 1;
FIG. 7 is a photomicrograph of an immunoblot showing the presence of hOP-1 in serum; and
FIG. 8A is a dose response curve for the induction of the 180 kDa and 140 kDa N-CAM isoforms in morphogen-treated NG108-15 cells;
FIG. 8B is a photomicrograph of a Western blot of whole cell extracts from morphogen-treated NG108-15 cells with an N-CAM-specific antibody; and
FIG. 9 (A and B) are photographs showing the effect of morphogen-specific antibody on mouse development (9B) compared to untreated, control mice (9A).
DETAILED DESCRIPTION OF THE INVENTION
It now has been discovered that the proteins described herein are found in nursing mother's milk and are useful as components of a dietary composition for enhancing tissue morphogenesis in a mammal, particularly in an individual at risk for normal tissue development and viability. As described herein, these proteins ("morphogens") are capable of enhancing tissue development in growing mammals, stimulating CAM expression and maintaining the normal tissue function in adult tissue.
Provided below are detailed descriptions of suitable morphogens useful in the compositions and methods of this invention, as well as methods for their administration and application, and numerous, nonlimiting examples which demonstrate the suitability of the morphogens described herein as active components of a dietary composition for a mammal; and 2) provide assays with which to test candidate morphogens for their efficacy. Specifically, examples are provided which (1) demonstrate the presence of endogenous morphogen in milk and human serum (Examples 1 and 2), (2) demonstrate the ability of morphogens to induce CAM expression in a mammal (Example 3), (3) demonstrate the ability of morphogens to enhance tissue development in developing embryos (Example 4) and juveniles (Example 5); (4) demonstrate the ability of morphogens to reduce an osteoporotic condition in a mammal (Example 6); (5) demonstrate the presence of morphogens in developing tissues and adult stomach and gut tissue, demonstrate the ability of parenterally provided morphogen to localize to stomach tissue, and describe protocols for identifying morphogen-synthesizing tissue (Example 7) and (6) describe protocols for obtaining morphogen-specific antibodies and measuring morphogens in solution (Example 8).
I. Useful Morphogens
As defined herein a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton or its functional equivalent (see supra). Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the "redifferentiation" of transformed cells. Details of how the morphogens useful in the method of this invention first were identified, as well as a description on how to make, use and test them for morphogenic activity are disclosed in U.S. Ser. No. 667,274 abandoned in favor of U.S. Ser. No. 752,764 (currently pending as U.S. Ser. No. 08/404,113), the disclosures of which
A candidate morphogen or morphogen composition can be evaluated for in vivo morphogenic utility generally according to the procedures set forth in U.S. Ser. No. 08/404,113. The proteins and compositions may be injected or surgically implanted in a mammal, following any of a number of procedures well known in the art. For example, surgical implant bioassays may be performed essentially following the procedure of Sampath et al. (1983) PNAS 80: 6591-6595.
Histological sectioning and staining is preferred to determine the extent of morphogenesis in vivo, particularly in tissue repair procedures. Excised implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 μm sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of the new tissue. Twelve day implants are usually sufficient to determine whether the implants contain newly induced tissue.
Successful implants exhibit a controlled progression through the stages of induced tissue development allowing one to identify and follow the tissue-specific events that occur. For example, in endochondral bone formation the stages include:
(1) leukocytes on day one;
(2) mesenchymal cell migration and proliferation on days two and three;
(3) chondrocyte appearance on days five and six;
(4) cartilage matrix formation on day seven;
(5) cartilage calcification on day eight;
(6) vascular invasion, appearance of osteoblasts, and formation of new bone on days nine and ten;
(7) appearance of osteoblastic and bone remodeling and dissolution of the implanted matrix on days twelve to eighteen; and
(8) hematopoietic bone marrow differentiation in the ossicle on day twenty-one.
In addition to histological evaluation, biological markers may be used as a marker for tissue morphogenesis. Useful markers include tissue-specific enzymes whose activity may be assayed (e.g., spectrophotometrically) after homogenization of the implant. These assays may be useful for quantitation and for obtaining an estimate of tissue formation quickly after the implants are removed from the animal. For example, alkaline phosphatase activity may be used as a marker for osteogenesis.
Incorporation of systemically provided morphogens may be followed using tagged morphogens (e.g., radioactively labelled) and determining their localization in new tissue, and/or by monitoring their disappearance from the circulatory system using a standard pulse-chase labeling protocol. The morphogen also may be provided with a tissue-specific molecular tag, whose uptake may be monitored and correlated with the concentration of morphogen provided. As an example, ovary removal in female rats results in reduced bone alkaline phosphatase activity, rendering the rats predisposed to osteoporosis. If the female rats now are provided with a morphogen, e.g., OP-1, a reduction in the systemic concentration of calcium (CA2+) is seen, which correlates with the presence of the provided morphogen and can be shown to correspond to increased alkaline phosphatase activity.
The morphogen to be assayed according to the above-described exemplary procedures can be purified from naturally-sourced material, or can be recombinantly produced from procaryotic or eucaryotic host cells, into which genetic material encoding a morphogen, e.g., genetic material bearing one of the nucleic acid sequences disclosed herein, has been introduced. Alternatively, the above-described exemplary procedures can be used to determine whether a novel protein suspected of being a morphogen indeed has morphogenic activity.
Particularly useful proteins include those which comprise the naturally derived sequences disclosed in Table II. Other useful sequences include biosynthetic constructs such as those disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).
Accordingly, the morphogens useful in the methods and compositions of this invention also may be described by morphogenically active proteins having amino acid sequences sharing 70% or, preferably, 80% homology (similarity) with any of the sequences described above, where "homology" is as defined herein above.
The morphogens useful in the method of this invention also can be described by any of the 6 generic sequences described herein ( Generic Sequences 1, 2, 3, 4, 5 and 6). Generic sequences 1 and 2 also may include, at their N-terminus, the sequence
______________________________________                                    
Cys     Xaa     Xaa     Xaa   Xaa   (Seq. ID No. 15)                      
1                             5                                           
______________________________________
Table II, set forth below, compares the amino acid sequences of the active regions of native proteins that have been id entified as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33), 60A protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP5 (Seq. ID No. 27) and BMP6 (Seq. ID No. 28). The sequences are aligned essentially following the method of Needleman et al. (1970) J. Mol. Biol., 48: 443-453, calculated using the Align Program (DNAstar, Inc.) In the table, three dots indicates that the amino acid in that position is the same as the amino acid in hOP-1. Three dashes indicates that no amino acid is present in that position, and are included for purposes of illustrating homologies. For example, amino acid residue 60 of CBMP-2A and CBMP-2B is "missing". Of course, both these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
                                  TABLE II                                
__________________________________________________________________________
hOP-1                                                                     
     Cys                                                                  
        Lys Lys His Glu Leu Tyr Val                                       
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
hOP-2                                                                     
     . . .                                                                
        Arg Arg . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
mOP-2                                                                     
     . . .                                                                
        Arg Arg . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
DPP  . . .                                                                
        Arg Arg . . .                                                     
                    Ser . . .                                             
                            . . .                                         
                                . . .                                     
Vgl  . . .                                                                
        . . .                                                             
            Lys Arg His . . .                                             
                            . . .                                         
                                . . .                                     
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Gly . . .                                             
                            . . .                                         
                                . . .                                     
CBMP-2A                                                                   
     . . .                                                                
        . . .                                                             
            Arg . . .                                                     
                    Pro . . .                                             
                            . . .                                         
                                . . .                                     
CBMP-2B                                                                   
     . . .                                                                
        Arg Arg . . .                                                     
                    Ser . . .                                             
                            . . .                                         
                                . . .                                     
BHP3 . . .                                                                
        Ala Arg Arg Tyr . . .                                             
                            Lys . . .                                     
GDF-1                                                                     
     . . .                                                                
        Arg Ala Arg Arg . . .                                             
                            . . .                                         
                                . . .                                     
60A  . . .                                                                
        Gln Met Glu Thr . . .                                             
                            . . .                                         
                                . . .                                     
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
BMP6 . . .                                                                
        Arg . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
     1              5                                                     
hOP-1                                                                     
     Ser                                                                  
        Phe Arg Asp Leu Gly Trp Gln Asp                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            Gln . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                Leu . . .                                 
mOP-2                                                                     
     Ser                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                Leu . . .                                 
DPP  Asp                                                                  
        . . .                                                             
            Ser . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                Asp . . .                                 
Vgl  Glu                                                                  
        . . .                                                             
            Lys . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                . . .                                     
                                    Asn                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            Gln . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2A                                                                   
     Asp                                                                  
        . . .                                                             
            Ser . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                Asn . . .                                 
CBMP-2B                                                                   
     Asp                                                                  
        . . .                                                             
            Ser . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                Asn . . .                                 
BHP3 Asp                                                                  
        . . .                                                             
            Ala . . .                                                     
                    Ile . . .                                             
                            . . .                                         
                                Ser Glu                                   
GDF-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                Glu Val . . .                                             
                            . . .                                         
                                His Arg                                   
60A  Asp                                                                  
        . . .                                                             
            Lys . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                His . . .                                 
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP6 . . .                                                                
        . . .                                                             
            Gln . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
        10                  15                                            
hOP-1                                                                     
     Trp                                                                  
        Ile Ile Ala Pro Glu Gly Tyr Ala                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        Val . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Gln . . .                                         
                                . . .                                     
                                    Ser                                   
mOP-2                                                                     
     . . .                                                                
        Val . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Gln . . .                                         
                                . . .                                     
                                    Ser                                   
DPP  . . .                                                                
        . . .                                                             
            Val . . .                                                     
                    . . .                                                 
                        Leu . . .                                         
                                . . .                                     
                                    Asp                                   
Vgl  . . .                                                                
        Val . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Gln . . .                                         
                                . . .                                     
                                    Met                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Lys . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2A                                                                   
     . . .                                                                
        . . .                                                             
            Val . . .                                                     
                    . . .                                                 
                        Pro . . .                                         
                                . . .                                     
                                    His                                   
CBMP-2B                                                                   
     . . .                                                                
        . . .                                                             
            Val . . .                                                     
                    . . .                                                 
                        Pro . . .                                         
                                . . .                                     
                                    Gln                                   
BHP3 . . .                                                                
        . . .                                                             
            . . .                                                         
                Ser . . .                                                 
                        Lys Ser Phe Asp                                   
GDF-1                                                                     
     . . .                                                                
        Val . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Arg . . .                                         
                                Phe Leu                                   
60A  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Gly                                   
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Lys . . .                                         
                                . . .                                     
                                    . . .                                 
            20                  25                                        
hOP-1                                                                     
     Ala                                                                  
        Tyr Tyr Cys Glu Gly Glu Cys Ala                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Ser                                   
mOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
DPP  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    His . . .                                             
                            Lys . . .                                     
                                    Pro                                   
Vgl  . . .                                                                
        Asn . . .                                                         
                . . .                                                     
                    Tyr . . .                                             
                            . . .                                         
                                . . .                                     
                                    Pro                                   
Vgr-1                                                                     
     . . .                                                                
        Asn . . .                                                         
                . . .                                                     
                    Asp . . .                                             
                            . . .                                         
                                . . .                                     
                                    Ser                                   
CBMP-2A                                                                   
     . . .                                                                
        Phe . . .                                                         
                . . .                                                     
                    His . . .                                             
                            Glu . . .                                     
                                    Pro                                   
CBMP-2B                                                                   
     . . .                                                                
        Phe . . .                                                         
                . . .                                                     
                    His . . .                                             
                            Asp . . .                                     
                                    Pro                                   
BMP3 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Ser . . .                                             
                            Ala . . .                                     
                                    Gln                                   
GDF-1                                                                     
     . . .                                                                
        Asn . . .                                                         
                . . .                                                     
                    Gln . . .                                             
                            Gln . . .                                     
                                    . . .                                 
60A  . . .                                                                
        Phe . . .                                                         
                . . .                                                     
                    Ser . . .                                             
                            . . .                                         
                                . . .                                     
                                    Asn                                   
BMP5 . . .                                                                
        Phe . . .                                                         
                . . .                                                     
                    Asp . . .                                             
                            . . .                                         
                                . . .                                     
                                    Ser                                   
BMP6 . . .                                                                
        Asn . . .                                                         
                . . .                                                     
                    Asp . . .                                             
                            . . .                                         
                                . . .                                     
                                    Ser                                   
                30                  35                                    
hOP-1                                                                     
     Phe                                                                  
        Pro Leu Asn Ser Tyr Met Asn Ala                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                Asp . . .                                                 
                        Cys . . .                                         
                                . . .                                     
                                    . . .                                 
mOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                Asp . . .                                                 
                        Cys . . .                                         
                                . . .                                     
                                    . . .                                 
DPP  . . .                                                                
        . . .                                                             
            . . .                                                         
                Ala Asp His Phe . . .                                     
                                    Ser                                   
Vgl  Tyr                                                                  
        . . .                                                             
            . . .                                                         
                Thr Glu Ile Leu . . .                                     
                                    Gly                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Ala His . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2A                                                                   
     . . .                                                                
        . . .                                                             
            . . .                                                         
                Ala Asp His Leu . . .                                     
                                    Ser                                   
CBMP-2B                                                                   
     . . .                                                                
        . . .                                                             
            . . .                                                         
                Ala Asp His Leu . . .                                     
                                    Ser                                   
GDF-1                                                                     
     Leu                                                                  
        . . .                                                             
            Val Ala Leu Ser Gly Ser**                                     
                                    . . .                                 
BMP3 . . .                                                                
        . . .                                                             
            Met Pro Lys Ser Leu Lys Pro                                   
60A  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Ala His . . .                                         
                                . . .                                     
                                    . . .                                 
BMP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Ala His Met . . .                                     
                                    . . .                                 
BMP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Ala His Met . . .                                     
                                    . . .                                 
                    40                                                    
hOP-1                                                                     
     Thr                                                                  
        Asn His Ala Ile Val Gln Thr Leu                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Leu . . .                                         
                                Ser . . .                                 
mOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Leu . . .                                         
                                Ser . . .                                 
DPP  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Val . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
Vgl  Ser                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Leu . . .                                         
                                . . .                                     
                                    . . .                                 
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2A                                                                   
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2B                                                                   
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP3 Ser                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Thr Ile . . .                                         
                                Ser Ile                                   
GDF-1                                                                     
     Leu                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Val Leu Arg Ala . . .                                 
60A  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
     45                 50                                                
hOP-1                                                                     
     Val                                                                  
        His Phe Ile Asn Pro Glu Thr Val                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            Asp . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        His Leu Met Lys . . .                                             
                            Asn Ala . . .                                 
mOP-2                                                                     
     . . .                                                                
        His Leu Met Lys . . .                                             
                            Asp Val . . .                                 
DPP  . . .                                                                
        Asn Asn Asn . . .                                                 
                        . . .                                             
                            Gly Lys . . .                                 
Vgl  . . .                                                                
        . . .                                                             
            Ser . . .                                                     
                    Glu . . .                                             
                            . . .                                         
                                Asp Ile                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            Val Met . . .                                                 
                        . . .                                             
                            . . .                                         
                                Tyr . . .                                 
CBMP-2A                                                                   
     . . .                                                                
        Asn Ser Val . . .                                                 
                        Ser --  Lys Ile                                   
CBMP-2B                                                                   
     . . .                                                                
        Asn Ser Val . . .                                                 
                        Ser --  Ser Ile                                   
BHP3 . . .                                                                
        Arg Ala**                                                         
                Gly Val Val Pro Gly Ile                                   
GDF-1                                                                     
     Met                                                                  
        . . .                                                             
            Ala Ala Ala . . .                                             
                            Gly Ala Ala                                   
60A  . . .                                                                
        . . .                                                             
            Leu Leu Glu . . .                                             
                            Lys Lys . . .                                 
BHP5 . . .                                                                
        . . .                                                             
            Leu Met Phe . . .                                             
                            Asp His . . .                                 
BHP6 . . .                                                                
        . . .                                                             
            Leu Met . . .                                                 
                        . . .                                             
                            . . .                                         
                                Tyr . . .                                 
        55                  60                                            
hOP-1                                                                     
     Pro                                                                  
        Lys Pro Cys Cys Ala Pro Thr Gln                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            Ala . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Lys                                   
mOP-2                                                                     
     . . .                                                                
        . . .                                                             
            Ala . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Lys                                   
DPP  . . .                                                                
        . . .                                                             
            Ala . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                . . .                                     
                                    . . .                                 
Vgl  . . .                                                                
        Leu . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                . . .                                     
                                    Lys                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Lys                                   
CBMP-2A                                                                   
     . . .                                                                
        . . .                                                             
            Ala . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                . . .                                     
                                    Glu                                   
CBMP-2B                                                                   
     . . .                                                                
        . . .                                                             
            Ala . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                . . .                                     
                                    Glu                                   
BMP3 . . .                                                                
        Glu . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                Glu Lys                                   
GDF-1                                                                     
     Asp                                                                  
        Leu . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Val . . .                                         
                                Ala Arg                                   
60A  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Arg                                   
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Lys                                   
BMP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Lys                                   
            65                  70                                        
hOP-1                                                                     
     Leu                                                                  
        Asn Ala Ile Ser Val Leu Tyr Phe                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        Ser . . .                                                         
                Thr . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Tyr                                   
mOP-2                                                                     
     . . .                                                                
        Ser . . .                                                         
                Thr . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Tyr                                   
Vgl  Met                                                                  
        Ser Pro . . .                                                     
                    . . .                                                 
                        Met . . .                                         
                                Phe Tyr                                   
Vgr-1                                                                     
     Val                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
DPP  . . .                                                                
        Asp Ser Val Ala Met . . .                                         
                                . . .                                     
                                    Leu                                   
CBMP-2A                                                                   
     . . .                                                                
        Ser . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Met . . .                                         
                                . . .                                     
                                    Leu                                   
CBMP-2B                                                                   
     . . .                                                                
        Ser . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Met . . .                                         
                                . . .                                     
                                    Leu                                   
BMP3 Met                                                                  
        Ser Ser Leu . . .                                                 
                        Ile . . .                                         
                                Phe Tyr                                   
GDF-1                                                                     
     . . .                                                                
        Ser Pro . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                Phe . . .                                 
60A  . . .                                                                
        Gly . . .                                                         
                Leu Pro . . .                                             
                            . . .                                         
                                . . .                                     
                                    His                                   
BMP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BMP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
                75                  80                                    
hOP-1                                                                     
     Asp                                                                  
        Asp Ser Ser Asn Val Ile Leu Lys                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
hOP-2                                                                     
     . . .                                                                
        Ser . . .                                                         
                Asn . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Arg                                   
mOP-2                                                                     
     . . .                                                                
        Ser . . .                                                         
                Asn . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    Arg                                   
DPP  Asn                                                                  
        . . .                                                             
            Gln . . .                                                     
                    Thr . . .                                             
                            Val . . .                                     
                                    . . .                                 
Vgl  . . .                                                                
        Asn Asn Asp . . .                                                 
                        . . .                                             
                            Val . . .                                     
                                    Arg                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            Asn . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
CBMP-2A                                                                   
     . . .                                                                
        Glu Asn Glu Lys . . .                                             
                            Val . . .                                     
                                    . . .                                 
CBMP-2B                                                                   
     . . .                                                                
        Glu Tyr Asp Lys . . .                                             
                            Val . . .                                     
                                    . . .                                 
BHP3 . . .                                                                
        Glu Asn Lys . . .                                                 
                        . . .                                             
                            Val . . .                                     
                                    . . .                                 
GDF-1                                                                     
     . . .                                                                
        Asn . . .                                                         
                Asp . . .                                                 
                        . . .                                             
                            Val . . .                                     
                                    Arg                                   
60A  Leu                                                                  
        Asn Asp Glu . . .                                                 
                        . . .                                             
                            Asn . . .                                     
                                    . . .                                 
BMP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
BHP6 . . .                                                                
        . . .                                                             
            Asn . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
                                    . . .                                 
                    85                                                    
hOP-1                                                                     
     Lys                                                                  
        Tyr Arg Asn Met Val Val Arg                                       
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
hOP-2                                                                     
     . . .                                                                
        His . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                Lys                                       
mOP-2                                                                     
     . . .                                                                
        His . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                Lys                                       
DPP  Asn                                                                  
        . . .                                                             
            Gln Glu . . .                                                 
                        Thr . . .                                         
                                Val                                       
Vgl  His                                                                  
        . . .                                                             
            Glu . . .                                                     
                    . . .                                                 
                        Ala . . .                                         
                                Asp                                       
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
CBMP-2A                                                                   
     Asn                                                                  
        . . .                                                             
            Gln Asp . . .                                                 
                        . . .                                             
                            . . .                                         
                                Glu                                       
CBMP-2B                                                                   
     Asn                                                                  
        . . .                                                             
            Gln Glu . . .                                                 
                        . . .                                             
                            . . .                                         
                                Glu                                       
BHP3 Val                                                                  
        . . .                                                             
            Pro . . .                                                     
                    . . .                                                 
                        Thr . . .                                         
                                Glu                                       
GDF-1                                                                     
     Gln                                                                  
        . . .                                                             
            Glu Asp . . .                                                 
                        . . .                                             
                            . . .                                         
                                Asp                                       
60A  . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        Ile . . .                                         
                                Lys                                       
BHP5 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
BMP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                Trp . . .                                                 
                        . . .                                             
                            . . .                                         
                                . . .                                     
     90                 95                                                
hOP-1                                                                     
     Ala                                                                  
        Cys Gly Cys His                                                   
mOP-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
hOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
mOP-2                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
DPP  Gly                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Arg                                                   
Vgl  Glu                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Arg                                                   
Vgr-1                                                                     
     . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
CBMP-2A                                                                   
     Gly                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Arg                                                   
CBMP-2B                                                                   
     Gly                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Arg                                                   
BHP3 Ser                                                                  
        . . .                                                             
            Ala . . .                                                     
                    Arg                                                   
GDF-1                                                                     
     Glu                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    Arg                                                   
60A  Ser                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
BMP5 Ser                                                                  
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
BMP6 . . .                                                                
        . . .                                                             
            . . .                                                         
                . . .                                                     
                    . . .                                                 
            100                                                           
__________________________________________________________________________
 **Between  residues  56 and 57 of BMP3 is a Val residue; between  residues  4
 and 44 of GDF1 lies the amino acid sequence GlyGly-Pro-Pro.
As is apparent from the foregoing amino acid sequence comparisons, significant amino acid changes can be made within the generic sequences while retaining the morphogenic activity. For example, while the GDF-1 protein sequence depicted in Table II shares only about 50% amino acid identity with the hOP1 sequence described therein, the GDF-1 sequence shares greater than 70% amino acid sequence homology (or "similarity") with the hOP1 sequence, where "homology" or "similarity" includes allowed conservative amino acid changes within the sequence as defined by Dayoff, et al., Atlas of Protein Sequence and Structure vol.5, supp.3, pp.345-362, (M. O. Dayoff, ed., Nat'l BioMed. Res. Fd'n, Washington D.C. 1979.)
The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in still another preferred aspect, the invention includes morphogens comprising species of polypeptide chains having the generic amino acid sequence referred to herein as "OPX", which defines the seven cysteine skeleton and accommodates the identities between the various identified mouse and human OP1 and OP2 proteins. OPX is presented in Seq. ID No. 29. As described therein, each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 16-23).
II. Formulations and Methods for Administering Therapeutic Agents
A. General Considerations
The morphogens may be provided to an individual by any suitable means, most preferably orally, or, alternatively, parenterally. Where the morphogen is to be provided parenterally, such as intravenously or by enteral feeding tube, the morphogen preferably comprises part of an aqueous solution. The solution is physiologically acceptable so that in addition to delivery of the desired morphogen to the patient, the solution does not otherwise adversely affect the patient's electrolyte and volume balance. The aqueous medium for the morphogen thus may comprise normal physiologic saline (0.85% NaCl, 0.15 M), pH 7-7.4. The aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents. One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA). The resultant solution preferably is vortexed extensively. If desired, a given morphogen may be made more soluble by association with a suitable molecule. For example, the pro form of the morphogenic protein comprises a species that is soluble in physiologically buffered solutions. In fact, the endogenous protein is thought to be transported (e.g., secreted and circulated) in this form. This soluble form of the protein may be obtained from the culture medium of morphogen-secreting mammalian cells. Alternatively, a soluble species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain. Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein, including salts, derivatives and analogs thereof. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 in physiologically buffered solutions by 80%. Other components found in milk and/or various serum proteins also may be useful.
Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990. Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, polybutyrate, tricalcium phosphate, lactide and lactide/glycolide copolymers, may be useful excipients to control the release of the morphogen in vivo.
As described above, the dietary supplements comprising the morphogens described herein preferably are provided orally. Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream. However, the morphogens described herein typically are acid stable and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.) In addition, at least one morphogen, OP-1, has been identified in mammary gland extract, colostrum and 57-day milk (see Example 1, below). Moreover, the OP-1 purified from mammary gland extract is morphogenically active. Specifically, this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen also is detected in the bloodstream (see Example 2, below). These findings indicate that oral and parenteral administration are viable means for administering morphogens to an individual. In addition, while the mature forms of certain morphogens described herein typically are sparingly soluble, the morphogen form found in milk (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.
The dietary compositions for oral administration may be formulated as a liquid, for example, as part of an aqueous medium as described above for parenteral administration, and which further may contain flavoring and coloring agents. The formulation also may be combined with a beverage or may be provided in a syrup. The dietary composition also may be provided as an aerosol for oral or nasal administration. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Alternatively, the dietary composition may be provided as a solid, for example as a tablet, capsule or lozenge. As for parenteral administration, formulations for oral administration also may include molecules to enhance a controlled release of the morphogen in vivo.
As will be appreciated by those skilled in the art, the concentration of the compounds described in a given dietary supplement composition will vary depending upon a number of factors, including the dosage number to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. The preferred dosage to be administered also is likely to depend on such variables as the type and extent of tissue development enhancement desired, the type and extent of any tissue damage present to be repaired, the overall health status of the particular individual, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration. In general terms, the compounds of this invention may be provided in a formulation containing about 0.001 to 10% w/v of morphogen to formulation. Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.1 μg/kg to 100 mg/kg of body weight per day. Optimally, the morphogen dosage given is between 0.1-100 μg of protein per kilogram weight of the individual. No obvious morphogen induced pathological lesions are induced when mature morphogen (e.g., OP-1, 20 μg) is administered daily to normal growing rats for 21 consecutive days. Moreover, 10 μg systemic injections of morphogen (e.g., OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.
In administering morphogens parenterally in the methods of the present invention, preferably a large volume loading dose is used at the start of the treatment. The treatment then is continued with a maintenance dose. In all cases administration dosages then can be monitored by measuring at intervals the levels of the morphogen in the blood.
B. Considerations for Infant and Other Formulas
1. Infant Formulas
In all cases the morphogens of this invention preferably are added to an infant formula that complies with the nutritional guidelines provided by the AAP and ESPGAN. Basic ingredients for infant formulas include cow's milk, protein, whey proteins, casein and its salts (i.e. calcium caseinate). Soy protein isolates may be substituted for milk-derived proteins, and preferably are used in the products made for infants with lactose intolerance and/or cow's protein intolerance. Protein hydrolyzates (i.e. casein and lactalbumin hydrolyzates) with low molecular weight, also may be used for these products.
The proportions of the diverse component nutrients preferably are similar to those of human milk. Thus, the ratio of whey proteins to casein preferably varies from 60:40 to 70:30 in infant formulas based on milk. The mixture of fats employed is made up of edible fats to provide an essential fatty acid profile. Lactose preferably is used as the carbohydrate source for at-term newborns infants, and dextrinmaltose preferably is employed in products used for the treatment of lactose intolerance and malabsorption syndromes in infancy.
Infant formulas according to the invention also preferably contain minerals (including calcium, phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese and iodine) and vitamins (including vitamin A, vitamin D3, vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E, vitamin K1, folic acid, biotin) adequate for the infants' requirements. Also, in the products whose source of proteins is derived from soy or protein isolates or hydrolyzates, carnitine preferably is included to satisfy the nutritional requirements for this compound in infants with malabsorptive syndromes.
A typical ready-to-feed morphogen-enriched formulation for infants, when diluted to feeding concentrations, preferably comprises in addition to the added morphogen, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.
A typical protein source for use in infant formula is electrodialyzed whey or electrodialyzed skim milk or milk whey, although other protein sources are also available and may be used. Preferred sugars include food grade substances such as glucose, dextrose, sucrose, or edible lactose. The following vitamins and minerals may also be incorporated in the infant formula: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, and B complex. These micronutrients are added in the form of commonly accepted nutritional compounds in amounts equivalent to those present in human milk on a per calories basis.
The infant formula according to the present invention also preferably is sterilized and subsequently used on a ready-to-feed basis, or can be stored as a concentrate. The concentrate can be prepared using standard procedures known in the art, and the formula can be reconstituted by rehydrating the concentrate. The infant formula preferably is a stable liquid and has a suitable shelf life. A more detailed description of infant formula considerations, including preferred formulations for newborn, preterm and low birth-weight infants, lactose-intolerant infants, may be found, for example, in U.S. Pat. No. 5,066,500 to Gil et al., the disclosure of which is incorporated herein by reference.
2. Other Nutritional Products
The morphogen-enriched dietary products for balanced nutrition (e.g., dietary food formulations) according to the present invention, preferably have, in addition to added morphogen, a composition of nutrients adequate to the specific requirements of not only healthy human in need of a balanced nutritional product, but also those individuals at risk for lost or reduced tissue function due malnutrition-maladsorption disorder, and/or altered metabolism. Individuals particularly affected by an altered metabolic function include postmenopausal women or aged individuals, hypercatabolic individuals, and individuals undergoing periods of rapid growth or physical stress, such as developing juveniles, and pregnant, lactating and nursing mothers. Other individuals at risk are those suffering from malnutrition, induced, for example, by starvation and/or an eating disorder, and individuals affected with energy-protein malnutrition and in hypercatablic states derived from traumatic, septic, surgical processes and other clinically-derived malabsorption syndromes.
Morphogen-enriched nutritional products according to the present invention preferably also provide mineral elements which include trace elements and vitamins in adequate proportions to satisfy the specific requirements of normal healthy individuals as well as individuals at risk, such as those suffering malabsorption-malnutrition processes and in a hypercatabolic state. The nutritional products also preferably are enriched with amino acids sources, vitamins, nucleosides and/or nucleotides in similar amounts to those present in ordinary foods.
As described above for infant formulas, liquid products may be formulated ready for consumption or as concentrates to be diluted before use. Preferably, liquid dietary compositions have pH values generally ranging from about 6.0 to about 8.0, most preferably 6.8-7.5.
Useful dietary compositions and considerations for their formulation are well described in the medical and nutritional arts. Useful compositions for clinical nutrition, also are described in detail in U.S. Pat. No. 5,066,500.
III. Examples
Example 1
Determination of the Presence of Morphogen in Milk
Morphogenically active OP-1 was demonstrated to be present in mammary gland extract, colostrum, and milk, as described below. The discovery that the morphogen naturally is present in milk, together with the known observation that mature, active OP-1 is acid-stable and protease-resistant, indicate that oral administration is a useful route for therapeutic administration of morphogen to a mammal. Oral administration typically is the preferred mode of delivery for extended or prophylactic therapies. In addition, the identification of morphogen in all milk forms, including colostrum, indicates that the protein plays a significant role in tissue development, including skeletal development of juveniles.
Rat mammary gland extract and bovine colostrum and 57 day milk were subjected to purification procedures designed to partially purify OP-1. The partially purified product then was examined for the presence of OP-1 by Western blot analysis using OP-1-specific antisera, and tested for in vivo and in vitro activity.
1.1 Purification
The purification protocol for all three "milk" forms (e.g., mammary gland extract, colostrum and 57-day milk), involved three chromatography steps: (1) cation-exchange chromatography (S-Sepharose and followed by Phenyl-Sepharose chromatography); (2) Copper-Immobilized Metal Affinity chromatography (Cu++-IMAC); and finally, (3) C-18 reverse phase chromatography. Fractions were sampled at each step for the presence of OP-1. Fraction samples for testing were dialyzed versus water/0.1% TFA, then against 30% acetonitrile/0.1% TFA for analysis on SDS-polyacrylamide gels and immunoblots, using standard methodologies well described in the art. Unless otherwise stated, the primary antibody used for the immunoblots was made against full length OP-1 produced in E.coli using standard recombinant DNA and antibody production techniques (see, for example, Example 8, below for a general description for producing morphogen-specific antibodies.) Fractions found to contain the morphogen then were applied to the next column step or used in the immunoreactivity or activity assays described below.
Essentially the same protocol was followed for all three milk sources, except that two alternative cation-exchange methodologies were employed for colostrum purification, described in detail below. Unless otherwise indicated, all chemicals referenced are standard, commercially available reagents, readily available from a number of sources, including Sigma Chemical, Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co., Milwaukee.
step 1. Cation-Exchange Chromatography
The S-Sepharose purification step was performed as follows. 200 ml of cation exchanger (S-Sepharose, Sigma Chemical Corp.) were equilibrated with equilibration buffer (6M urea, 20 mM MES, 70 mM NaCl, pH 6.5). The supernatant from the centrifuged extract was diluted to final concentration of 6M urea, 20 mM MES, 70 mM NaCl, pH 6.5. After loading, the column was washed to baseline using equilibration buffer, and the bound components were eluted stepwise from the column with 6M urea, 20 mM MES, 100 mM and 500 mM NaCl, pH 6.5. The more tightly bound components then were eluted with 4M guanidine, 20 mM sodium phosphate, pH 7.0.
The Phenyl-Sepharose purification step was performed as follows. 15 ml of Phenyl-Sepharose CL-4B (Sigma) were equilibrated with 6M urea, 20 mM HEPES, 1M ammonium sulfate, 300 mM NaCl, pH 7.0. The 500 mM NaCl eluate from the S-Sepharose step was diluted with 6M urea, 20 mM HEPES, 3M ammonium sulfate, 300 mM NaCl, pH 7.0, to a final concentration of 1M ammonium sulfate, pH 7.0. After loading, the column was washed to baseline with equilibration buffer. The column was eluted with 6M urea, 20 mM HEPES, 0.6M ammonium sulfate, 300 mM NaCl, pH 7.0, and then with 4M guanidine, 20 mM sodium phosphate, pH 7.0.
Two alternative cation-exchange chromatography schemes (A and B) were exploited in the purification of OP-1 from colostrum, as follows. For both schemes, 200 ml of S-Sepharose (Sigma) was poured into a 5×10 cm Bio-Rad econocolumn (Bio-Rad, Inc. Cambridge.)
Scheme A: The colostrum, which had been diluted to 6M urea, 20 mM sodium phosphate, pH 7.0, was loaded onto a column equilibrated with 6M urea, 20 mM sodium phosphate, 50 mM NaCl, pH 7.0. Elution was stepwise, with 6M urea, 20 mM sodium phosphate, 100 mM and then 500 mM NaCl, pH 7.0; and the final wash was with 4M guanidine, 20 mM sodium phosphate, pH 7.0. The Phenyl-Sepharose column was run as described above, except that sodium phosphate was used as the running buffer instead of HEPES. The Phenyl-Sepharose bound fraction (0.0M ammonium sulfate eluate) from scheme A then was dialyzed into 6M urea, 20 mM Hepes, 500 mM NaCl, pH 7.0, before it was applied to the Cu++-IMAC column, which was run as described below.
Scheme B: The alternative S-Sepharose purification was performed as follows. Ethanol-precipitated protein was loaded onto an S-Sepharose column equilibrated in 6M urea, 20 mM MES, 50 mM NaCl, pH 6.5. Elution was stepwise with 6M urea, 20 mM MES, 100 mM NaCl and then 500 mM NaCl, and finally 4M guanidine, 20 mM sodium phosphate, pH 7.0. The Phenyl-Sepharose column was run as described above, with the 0.0M ammonium sulfate eluate then applied to a Cu++-IMAC column.
step 2. Cu++IMAC Chromatography
The Cu++IMAC purification step was performed as follows. 10 ml of Pharmacia Fast Flow Chelating Resin were charged with 0.2M cupric sulfate, and equilibrated with 6M urea, 20 mM HEPES, 0.5M NaCl, pH 7.0. After loading, the column was washed to baseline with equilibration buffer. Elution from the column was stepwise, using equilibration buffer containing 1 mM, 5 mM, or 10 mM imidazole. The column then was stripped with equilibration buffer containing 10 mM EDTA. The 10 mM imidazole elution was dialyzed against water/0.1% TFA, then against 30% acetonitirile/0.1% TFA.
step 3. Reverse Phase Chromatography
The C-18 reverse phase chromatography purification step was performed as follows. An HPLC C-18 semi-prep column was used for the final purification step. The gradient used was 30-50% acetonitrile/0.1% TFA over 60 minutes at 3 ml/minutes. After the sample was loaded, the column was washed to baseline with 30% acetonitrile/0.1% TFA before the gradient is started. Fractions collected were 3 ml in size. Chromatograms were read at 214 nm.
(a) OP-1 from Rat Mammary Gland Extract
Mammary glands were obtained from 2 female Long Evan rats (Charles River Labs, Wilmington, Mass.) one week post-partum. The excised glands were mildly homogenized in 6M urea, 20 mM methylethansulfonate (MES), 0.5M NaCl, pH 6.5 using a polytron homogenizer. The suspension then was centrifuged for 20 minutes at 8,000 RPM, and the supernatant removed for further purification.
Following S-Sepharose chromatography, fractions containing 6M urea, 20 mM MES containing 500 mM NaCl, also appeared to contain OP-1 as determined by SDS and immunoblot, and were applied to the Phenyl-Sepharose column. The eluate from the 6M urea, 20 mM HEPES, 300 mM NaCl, pH 7.0 elution step from this column were found to contain OP-1. This eluate then was applied to a Cu++-IMAC column. Eluate fractions found to contain OP-1 were then applied to the C-18 column and chromatographed as described.
FIG. 1(A) shows the chromatogram and 1(B) the corresponding Western blot for fractions from the C-18 reverse phase chromatography step run under reducing conditions. Lane S of the Western blot is a standard, containing reduced, purified, recombinantly-produced OP-1. The arrows show molecular weight markers corresponding to 17, 27, and 39 Kd. The reduced monomer run at approximately 16-18 Kd; the oxidized homodimer at approximately 36 Kd. Lanes 13-30 represent the corresponding fractions of the C-18 reverse phase column as numbered in FIG. 1(A). As can be seen in FIG. 1(B), mammary extract OP-1 elutes primarily in fractions 21-25 from this final chromatography step.
(b) OP-1 from Bovine Colostrum
Colostrum is the first milk to be produced by the mother following birth. Approximately 5 gallons of bovine colostrum were obtained from a local dairy farm and delipidated by centrifugation (8000 rpm for approximately 10 min. at 4° C.). The supernatant then was filtered through cheese cloth. The filtered supernatant was stored in 500 ml aliquots at 70° C.
50 ml of colostrum were diluted with 100 ml of 9M urea, 30 mM sodium phosphate, pH 7.0. Alternately, 50 ml of colostrum was added to 50 ml of 8M guanidine-HCl, 50 mM Tris, pH 7.2 and precipitated with 40%, then 85% ice cold ethanol. The pellet was washed with 90% cold ethanol and lyophilized overnight. The lyophilized pellet was resuspended in 6M urea, 20 mM MES, 500 mM NaCl, pH 6.5, stirred overnight at 4° C., and centrifuged at 9,000 RPM for 10 minutes to clarify the suspension before loading onto the column as described in schemes A and B, above.
Following S-Sepharose chromatography by scheme A, both the 100 mM and the 500 mM eluate fractions were found to contain OP-1, with the 100 mM fraction containing relatively more morphogen. This fraction then was loaded onto the Phenyl-Sepharose column following dilution with an equal volume of 6M urea, 20 mM sodium phosphate, 2M ammonium sulfate, and 300 mM NaCl.
Following S-Sepharose chromatography by scheme B, the 500 mM NaCl eluate was found to contain OP-1 and was loaded onto a Phenyl-Sepharose column as described above, following dilution with 6M urea, 40 mM HEPES, 2M ammonium sulfate, pH 7.0.
Following Cu++IMAC chromatography OP-1 was identified in the 5 mM and 10 mM imidazole eluates for both purification schemes, and was dialyzed for further purification on the C-18 column.
Both purification schemes produce purified OP-1, as determined by immunoblot. FIG. 2 shows the chromatogram (A) and corresponding Western blot (B) for results of purification scheme A (FIG. 2B-1, reduced and FIG. 2B-2, oxidized); and FIG. 3 shows the chromatogram (A) and Western blot (B, reduced) for C-18-purified protein from scheme B. As for FIG. 1B, lane S in FIGS. 2B and 3B is a standard, containing purified, recombinantly produced OP-1; 17, 27 and 39 are molecular weight markers, and lane numbers correspond to fraction numbers in the corresponding chromatograms. OP-1 purified by scheme A appears predominantly in fractions 18-27, and OP-1 purified by scheme B appears predominantly in fractions 18-25.
OP-1 from 57-day milk
Milk was obtained from the same cow from which the colostrum came, 57 days after the birth of the calf. The milk was delipidated by centrifugation at 10,000 RPM for 15 minutes, and the milk was poured off and away from the fat layer.
100 ml of milk then were diluted with 200 ml of 9M urea, 30 mM MES, pH 6.5 and loaded onto a 200 ml S-Sepharose column which had been equilibrated with 6M urea, 20 mM MES, 50 mM NaCl, pH 6.5. Elution was with 6M urea, 20 mM MES, 100 mM and 500 mM NaCl, and 4M guanidine, 20 mM sodium phosphate, pH 7.0. The 500 mM elution was put over a Phenyl-Sepharose column after being diluted with an equal volume of 6M urea, 20 mM MES, 2M ammonium sulfate, pH 7.0.
The Phenyl-Sepharose column then was run as described above. The Phenyl-Sepharose-bound sample was eluted and applied to a Cu++IMAC column, prepared and run as described above. The 10 mM imidazole eluate was found to contain OP-1 and was dialyzed for further purification on the C-18 column.
The C-18 reverse phase chromatography column and gradient were performed as described above. The results are presented in FIG. 4A (chromatogram) and 4B (immunoblot, 10B-1, oxidized; 4B-2, reduced.) As above, lane S is a standard, containing purified, recombinantly produced OP-1; 17, 27, and 39 are molecular weight markers, and the lane numbers correspond to the fractions numbers in FIG. 4A. OP-1 purified from 57-day milk appears predominantly in fractions 18-26.
1.2 OP-1 Characterization by immunoreactivity
OP-1 purified from the different milk sources as described above also were characterized by Western blotting using antibodies raised against OP-1 and BMP2. Antibodies were prepared using standard immunology protocols well known in the art, and as described in Example 8, below using full-length E. coli-produced OP-1 and BMP2 as the immunogens.
As shown in FIG. 5 OP-1 purified from colostrum reacts with the anti-OP-1 antibody, but not with anti-BMP2 antibody. In FIG. 5A and B, lane 1 contains reduced, purified, recombinantly-produced OP-1; lane 2 contains C-18 purified bovine colostrum, and lane 3 contains reduced COP-16, a biosynthetic construct having morphogenic activity and an amino acid sequence modeled on the proteins described herein, but having highest amino acid sequence homology with BMP2 (see U.S. Pat. No. 5,011,691 for the COP-16 amino acid sequence.) In FIG. 5A the gel was probed with anti-OP-1 antibody; in FIG. 5B, the gel was probed with anti-BMP2 antibody. As can be seen in the figure, anti-OP-1 antibody hybridizes with protein in lanes 1 and 2, but not 3; while anti-BMP2 antibody hybridizes with lane 3 only.
C-18 purified mammary gland extract and 57-day milk also were shown to react with anti-OP-1 antibodies, including antibody raised against the full length E. coli OP-1, full length mammalian-produced OP-1, and the OP-1 Ser-17-Cys peptide (e.g., the OP-1 N-terminal 17 amino acids).
1.3 OP-1 Characterization by Activity
The morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo as follows. 33% of each OP-1 immunoreactive fraction of C-18-purified mammary gland extract was lyophilized and resuspended in 220 μl of 50% acetonitrile/0.1% TFA. After vortexing, 25 mg of collagen matrix was added. The samples were lyophilized overnight, and implanted in Long Evans rats (Charles River Laboratories, Wilmington, Mass., 28-35 days old). Each fraction was implanted in duplicate. For details of the collagen matrix implantation procedure, see, for example, U.S. Pat. No. 4,968,590, hereby incorporated by reference. After 12 days, the implants were removed and evaluated for new bone formation by histological observation.
The results are presented in FIG. 6A, where "% activity" refers to the % of bone formation/total area covered by bone in the histology sample. In the figure, solid bars represent implants using mammary extract-derived OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see FIG. 1B), and the hatched bar represents implants using recombinantly produced OP-1 (600 ng). The results demonstrate that the peak bone forming activity of C-18-purified mammary gland extract corresponds with the immunoreactive fraction peaks of FIG. 1B (compare FIG. 6A and 1B.)
Similarly, the morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vitro by measuring alkaline phosphatase activity in vitro using the following assay. Test samples were prepared using 15-20% of individual immunoreactive fractions from the C-18 run which were precipitated and resuspended in a smaller volume of 50% acetonitrile/0.1% TFA. Alkaline phosphatase activity was tested using ROS 17/2.8 cells (Rat Osteosarcoma, e.g., obtained, for example, from Dr. Robert J. Majeska, Mt. Sinai Medical Center, New York, N.Y., in a standard alkaline phosphatase activity assay (see, for example, U.S. Pat. No. 4,968,590). The results, presented in FIG. 6B, indicate that the immunoreactive fractions obtained from C-18-purified mammary gland extract correspond with alkaline phosphatase activity in vitro (compare FIG. 6B and FIG. 1B.) In FIG. 6B solid bars represent assays performed with mammary gland-purified OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see FIG. 1B), the hatched bar represents the assay performed with purified, recombinantly-produced OP-1 (100 ng/ml), and the cross-hatched bar represents background. As for FIG. 6A, alkaline phosphatase activity corresponds with immunoreactivity of the C-18-purified extract (compare FIG. 6B and 1B.)
Example 2.
Morphogen Identification in Human Serum
OP-1 was detected in human serum using the following assay. A monoclonal antibody raised against mammalian, recombinantly produced OP-1 using standard immunology techniques well described in the art and described generally in Example 8, was immobilized by passing the antibody over an activated agarose gel (e.g., Affi-Gel™, from Bio-Rad Laboratories, Richmond, Calif., prepared following manufacturer's instructions), and used to purify OP-1 from serum. Human serum then was passed over the column and eluted with 3M K-thiocyanate. K-thiocyanante fractions then were dialyzed in 6M urea, 20 mM PO4, pH 7.0, applied to a C8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient. Mature, recombinantly produced OP-1 homodimers elute between 20-22 minutes. Fractions then were collected and tested for the presence of OP-1 by standard immunoblot. FIG. 7 is an immunoblot showing OP-1 in human sera under reducing and oxidized conditions. In the figure, lanes 1 and 4 are OP-1 standards, run under oxidized (lane 1) and reduced (lane 4) conditions. Lane 5 shows molecular weight markers at 17, 27 and 39 kDa. Lanes 2 and 3 are human sera OP-1, run under oxidized (lane 2) and reduced (lane 3) conditions.
Example 3.
Morphogen-Induced CAM Expression
The morphogens described herein induce CAM expression as part of their induction of morphogenesis. CAMs are morphoregulatory molecules identified in all tissues as an essential step in tissue development. N-CAMs, which comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where "180", "140" and "120" indicate the apparent molecular weights of the isoforms as measured by polyacrylamide gel electrophoresis) are expressed at least transiently in developing tissues, and permanently in nerve tissue. Both the N-CAM-180 and N-CAM-140 isoforms are expressed in both developing and adult tissue. The N-CAM-120 isoform is found only in adult tissue. Another neural CAM is L1.
CAMs are implicated in normal tissue development; N-CAMs are implicated in appropriate neural development, including appropriate neurulation, neuronal migration, fasciculation, and synaptogenesis. Inhibition of N-CAM production, as by complexing the molecule with an N-CAM-specific antibody, inhibits retina organization, including retinal axon migration, and axon regeneration in the peripheral nervous system, as well as axon synapses with target muscle cells. CAMs also have been postulated as part of a morphoregulatory pathway whose activity is induced by a to date unidentified molecule (See, for example, Edelman, G. M. (1986) Ann. Rev. Cell Biol. 2: 81-116). Without being limited to any given theory, the morphogens described herein may act as the inducer of this pathway.
The morphogens described herein can stimulate CAM production. As described below, the morphogens stimulate L1 and N-CAM production, including all three isoforms of the N-CAM molecule, in nerve tissue. A detailed description of this protocol is described in copending U.S. Ser. No. 922,813 the disclosure of which is incorporated hereinabove by reference.
In this example NG108-15 cells were cultured for 4 days in the presence of increasing concentrations of OP-1 and standard Western blots performed on whole cells extracts. The NG108-15 cell line is a hybrid cell line (neuroblastoma x glioma, American Type Culture Collection, Rockville, Md.). N-CAM isoforms were detected with an antibody which crossreacts with all three isoforms, mAb H28.123, obtained from Sigma Chemical Co., St. Louis, the different isoforms being distinguishable by their different mobilities on an electrophoresis gel. Control NG108-15 cells (untreated) express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined by western blot analyses using up to 100 μg of protein. As shown in FIG. 8, treatment of NG108-15 cells with OP-1 resulted in a dose-dependent increase in the expression of the 180 kDa and 140 kDa isoforms, as well as the induction of the 120 kDa isoform. FIG. 8B is a Western blot of OP1-treated NG108-15 cell extracts, probed with mAb H28.123, showing the induction of all three isoforms. FIG. 8A is a dose response curve of N-CAM-180 and N-CAM-140 induction as a function of morphogen concentration. N-CAM-120 is not shown in the graph as it could not be quantitated in control cells. However, as is clearly evident from the Western blot in FIG. 8A, N-CAM-120 is induced in response to morphogen treatment. The induction of the 120 isoform also indicates that morphogen-induced redifferentiation of transformed cells stimulates not only redifferentiation of these cells from a transformed phenotype, but also differentiation to a phenotype corresponding to a developed cell. The differential induction of N- CAM 180 and 140 isoforms seen may be because constitutive expression of the 140 isoform is close to maximum. In addition, the increase in N-CAM expression corresponded in a dose-dependent manner with the morphogen induction of multicellular aggregates.
In addition, the cell aggregation effects of OP-1 on NG108-15 cells can be inhibited with anti-N-CAM antibodies or antisense N-CAM oligonucleotides. Antisense oligonucleotides can be made synthetically on a nucleotide synthesizer, using standard means known in the art. Preferably, phosphorothioate oligonucleotides ("S-oligos") are prepared, to enhance transport of the nucleotides across cell membranes. Concentrations of both N-CAM antibodies and N-CAM antisense oligonucleotides sufficient to inhibit N-CAM induction also inhibited formation of multilayered cell aggregates. Specifically, incubation of morphogen-treated NG108-115 cells with 0.3-3 μM N-CAM antisense S-oligos, 5-500 μM unmodified N-CAM antisense oligos, or 10 μg/ml mAb H28.123 significantly inhibits cell aggregation. It is likely that morphogen treatment also stimulates other CAMs, as inhibition is not complete.
The experiments also have been performed with soluble morphogen (e.g., mature OP-1 associated with its pro domain) which also specifically induced CAM expression.
Example 4.
Effect of Morphogen Neutralization on Embryogenesis
As described in Example 7, below, at least one morphogen, OP2, is found principally in early developing embryos (8-day embryos). As described below, morphogen neutralization with morphogen-specific antibodies inhibits embryogenesis.
Morphogen inhibition in developing embryos inhibits tissue and organ development. Specifically, 9-day mouse embryo cells, cultured in vitro under standard culturing conditions, were incubated in the presence and absence of an OP-1-specific monoclonal antibody prepared using recombinantly produced, purified mature OP-1 as the immunogen. The antibody was prepared using standard antibody production means well known in the art and essentially as described for Example 9, below. After two days, the effect of the antibody on the developing embryo was evaluated by histology using standard histology procedures well known in the art. As determined by histological examination, the OP-1-specific antibody specifically inhibits eye lobe formation in the developing embryo. In particular, the diencephalon outgrowth does not develop. In addition, the heart is malformed. Moreover, in separate immunolocalization studies on embryo sections with labelled OP-1 specific antibody, the OP-1-specific antibody localizes to neural epithelia.
Similarly, morphogen activity may be demonstrated in fetal development in the mouse model using the following assay. Single lip injections comprising 10-100 μg/injection of morphogen-specific antibody are administered to pregnant female mice during each day of the gestation period and tissue development (e.g., bone development) in treated and control new mice evaluated by standard histomorphometric analysis at birth.
Finally, stimulation of endogenous morphogen antibody production in egg-laying hens interferes with shell formation in the developing eggs.
All of these data demonstrate that inhibition of morphogen activity significantly interferes with tissue development during embryogenesis.
Example 5.
Effect of Morphogen Neutralization on Juvenile Tissue Development
The effect of the morphogens described herein on tissue development in developing mammals also may be demonstrated using neutralizing antibodies specific for particular morphogens and assessing the effect of these antibodies on tissue development as described below. Specifically, anti-morphogen monoclonal and/or polyclonal antibodies may be prepared using standard methodologies including, for example, the protocol provided in Example 8, below, and provided to juveniles to inhibit the activity of endogenous morphogens.
Generally, purified antibodies are provided regularly to new born mice, e.g., 10-100 μg/injection/day for 10-15 days. At 10 or 21 days, the mice are sacrificed and the effect of morphogen on bone development assessed by body weight, gross visual examination and histology. In this example, anti-OP-1 antibodies were used in 10 μg injections/day for 14 days, and the mice were sacrificed at 21 days. As is dramatically demonstrated in FIG. 9, mice treated with OP-1 specific antibody show consistent and significant stunted growth, including reduced body length and body weight, (9B) as compared with untreated mice (9A). Histological examination showed reduced bone growth as evidenced by reduced bone size in the treated mice.
In a variation on this protocol, single lip injections also may be provided to older juveniles and adult mice (e.g., 10-100 μg) over a prolonged time (e.g., 10-15 days) to evaluate the effect or morphogen neutralization on bone growth and bone integrity and to evaluate the onset of osteoporosis.
Example 6.
Morphogen Treatment of Osteoporosis
6.1 Effect of Morphogen on Trabecular Bone in Ovariectomized (OVX) Rats
Aged individuals, and particularly postmenopausal women are particularly at risk for osteoporosis. Provided below is an animal osteoporosis model demonstrating the ability of morphogens to substantially inhibit and/or reduce the tissue damage effects associated with osteoporosis, wherein osteoporosis is induced by ovary removal in rats. Bone growth is evaluated in these animals by measuring serum alkaline phosphatase and osteocalcin levels in treated and untreated rats.
Forty Long-Evans rats (Charles River Laboratories, Wilmington) weighing about 200 g each are ovariectomized (OVX) using standard surgical procedures, and ten rats are sham-operated. The ovariectomization of the rats produces an osteoporotic condition within the rats as a result of decreased estrogen production. Food and water are provided ad libitum. Eight days after ovariectomy, the rats, prepared as described above, were divided into five groups: (A), 10 sham-operated rats; (B), 10 ovariectomized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the tail vein; (C) 10 ovariectomized rats receiving about 1 mg of 17βE2 (17-β-estradiol E2) by intravenous injection through the tail vein; (D) 9 ovariectomized rats receiving daily injections of approximately 2 μg of morphogen by tail vein for 22 days; and (E) 9 ovariectomized rats receiving daily injections of approximately 20 μg of morphogen by tail vein for 22 days. In this example, OP-1 was the morphogen tested.
On the 15th and 21st day of the study, each rat was injected with 5 mg of tetracycline, and on day 22, the rats were sacrificed. The body weights, uterine weights, serum alkaline phosphate levels, serum calcium levels and serum osteocalcin levels then were determined for each rat. The results are shown in Tables III and IV.
              TABLE III                                                   
______________________________________                                    
Body Weights, Uterine Weights and Alkaline Phosphatase                    
          Body Weights                                                    
                     Uterine Weights                                      
                                 Alk. Phosphatase                         
Group     (g)        (g)         (U/L)                                    
______________________________________                                    
A-SHAM    250.90 ± 17.04                                               
                     0.4192 ± 0.10                                     
                                 43.25 ± 6.11                          
B-OVX + PBS                                                               
          273.40 ± 16.81                                               
                     0.1650 ± 0.04                                     
                                 56.22 ± 6.21                          
C-OVX + E2                                                                
          241.66 ± 21.54                                               
                     0.3081 ± 0.03                                     
                                 62.66 ± 4.11                          
D-OVX + OP-1                                                              
          266.67 ± 10.43                                               
                     0.1416 ± 0.03                                     
                                 58.09 ± 12.97                         
(2 μg)                                                                 
E-OVX + OP-1                                                              
          272.40 ± 20.48                                               
                     0.1481 ± 0.05                                     
                                 66.24 ± 15.74                         
(20 μg)                                                                
______________________________________                                    

              TABLE IV                                                    
______________________________________                                    
Serum Calcium and Serum Osteocalcin Levels                                
            Serum Calcium Serum Osteocalcin                               
Group       (ng/dl)       (ng/ml)                                         
______________________________________                                    
A-SHAM      8.82 ± 1.65                                                
                           64.66 ± 14.77                               
B-OVX + PBS 8.95 ± 1.25                                                
                           69.01 ± 10.20                               
C-OVX + E2  9.20 ± 1.39                                                
                           67.13 ± 17.33                               
D-OVX + OP-1                                                              
            8.77 ± 0.95                                                
                          148.50 ± 84.11                               
(2 μg)                                                                 
E-OVX + OP-1                                                              
            8.67 ± 1.94                                                
                          182.42 ± 52.11                               
(20 μg)                                                                
______________________________________
The results presented in Table III and IV show that intravenous injection of morphogen into ovariectomized rats produces a significant increase in serum alkaline phosphatase and serum osteocalcin levels and demonstrates that systemic administration of the morphogen stimulates bone formation in osteoporotic bone.
6.2 Histomorphometric Analysis of Morphogen on the Tibia Diaphysis in Ovariectomized(OVX) Rats
Fifteen female Long-Evans rats weighing about 160 g were ovariectomized (OVX) to produce an osteoporotic condition and five rats were sham operated (Charles River Laboratories, Wilmington, Mass.) as described for Example 8. Food and water were provided ad libitum. Twenty-two days after ovariectomy, the rats were divided into four groups: (A) sham-operated (1 ml of PBS by intravenous injection through tail vein (5 rats); (B) OVX, into which nothing was injected (5 rats); (C) OVX, receiving about 1 mg of 17βE2 by intravenous injection through the tail vein (5 rats), and (D) OVX, receiving about 1 βg of morphogen by intravenous injection through the tail vein (5 rats). In this example, OP-1 was morphogen tested.
The rats were injected daily as described for seven days, except no injections were given on the thirteenth day. The rats then were sacrificed on the nineteenth day. The tibial diaphyseal long bones then were removed and fixed in ethanol and histomorphometric analysis was carried out using standard procedures well known in the art. The results are shown in Table V.
                                  TABLE V                                 
__________________________________________________________________________
           (A)    (B)   (C)    (D)                                        
MEASUREMENT                                                               
           CONTROL                                                        
                  OVX   OVX + E.sub.2                                     
                               OVX + OP-1                                 
__________________________________________________________________________
Longitudinal Growth                                                       
           20.2 ± 0.3                                                  
                  19.4 ± 0.2                                           
                         4.9 ± 0.5                                     
                               17.9 ÷ 0.9                             
Rate (μm/day)                                                          
Cancellous Bone                                                           
           20.2 ± 1.5                                                  
                  13.0 ± 1.6                                           
                        13.7 ± 2.1                                     
                               16.6 ± 1.8                              
Volume (BV/TV,                                                            
bone vol/total vol)                                                       
Cancellous Bone                                                           
           16.2 ± 1.8                                                  
                   9.6 ± 0.9                                           
                        11.5 ± 1.1                                     
                               12.2 ± 0.7                              
Perimeter (mm)                                                            
Labeled Cancellous                                                        
           35.5 ± 1.5                                                  
                  51.9 ± 5.6                                           
                        58.0 ± 4.2                                     
                               39.2 ± 1.9                              
Perimeter (%)                                                             
Mineral Apposition                                                        
            1.76 ± 0.14                                                
                   2.25 ± 0.16                                         
                         1.87 ± 0.08                                   
                                1.86 ± 0.20                            
Rate (μm/day)                                                          
__________________________________________________________________________
The results presented in Table V confirm the results of Example 6.1, namely that intravenous injection of OP-1 into ovariectomized rats stimulates bone growth for bone which had been lost due to the drop in estrogen within the individual rat. Specifically, the inhibition of cancellous bone volume in OVX rats is repaired by the systemically provided morphogen. In addition, in morphogen-treated rats the labelled cancellous perimeter and mineral apposition rate now return to levels measured in the control, sham-operated rats. Moreover, morphogen treatment does not inhibit longitudinal bone growth, unlike estrogen treatment, which appears to inhibit bone growth significantly. Accordingly, systemic administration of a morphogen in therapeutically effective concentrations effectively inhibits loss of bone mass in a mammal without inhibiting natural bone formation.
Example 7.
Identification of Morphogen-Expressing Tissue
Determining the tissue distribution of morphogens may be used to identify different morphogens expressed in a given tissue, as well as to identify new, related morphogens. Tissue distribution also may be used to identify useful morphogen-producing tissue for use in screening and identifying candidate morphogen-stimulating agents. The morphogens (or their mRNA transcripts) readily are identified in different tissues using standard methodologies and minor modifications thereof in tissues where expression may be low. For example, protein distribution may be determined using standard Western blot analysis or immunofluorescent techniques, and antibodies specific to the morphogen or morphogens of interest. Similarly, the distribution of morphogen transcripts may be determined using standard Northern hybridization protocols and transcript-specific probes.
Any probe capable of hybridizing specifically to a transcript, and distinguishing the transcript of interest from other, related transcripts may be used. Because the morphogens described herein share such high sequence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe specific for the pro region of the immature protein and/or the N-terminal region of the mature protein. Another useful sequence is the 3' non-coding region flanking and immediately following the stop codon. These portions of the sequence vary substantially among the morphogens of this invention, and accordingly, are specific for each protein. For example, a particularly useful Vgr-1-specific probe sequence is the PvuII-SacI fragment, a 265 bp fragment encoding both a portion of the untranslated pro region and the N-terminus of the mature sequence (see Lyons et al. (1989) PNAS 86: 4554-4558 for a description of the cDNA sequence). Similarly, particularly useful mOP-1-specific probe sequences are the BstX1l-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb sequence immediately upstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an 0.3 Kb fragment containing a portion of the 3'untranslated sequence (See Seq. ID No. 18, where the pro region is defined essentially by residues 30-291.) Similar approaches may be used, for example, with hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and 22.)
Using these morphogen-specific probes, which may be synthetically engineered or obtained from cloned sequences, morphogen transcripts can be identified in mammalian tissue, using standard methodologies well known to those having ordinary skill in the art. Briefly, total RNA is prepared from various adult murine tissues (e.g., liver, kidney, testis, heart, brain, thymus and stomach) by a standard methodology such as by the method of Chomczyaski et al. ((1987) Anal. Biochem 162: 156-159) and described below. Poly (A)+RNA is prepared by using oligo (dT)-cellulose chromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.). Poly (A)+RNA (generally 15 μg) from each tissue is fractionated on a 1% agarose/formaldehyde gel and transferred onto a Nytran membrane (Schleicher & Schuell). Following the transfer, the membrane is baked at 80° C. and the RNA is cross-linked under UV light (generally 30 seconds at 1 mW/cm2). Prior to hybridization, the appropriate probe is denatured by heating. The hybridization is carried out in a lucite cylinder rotating in a roller bottle apparatus at approximately 1 rev/min for approximately 15 hours at 37° C. using a hybridization mix of 40% formamide, 5×Denhardts, 5×SSPE, and 0.1% SDS. Following hybridization, the non-specific counts are washed off the filters in 0.1×SSPE, 0.1% SDS at 50° C.
Examples demonstrating the tissue distribution of various morphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2 in developing and adult tissue are disclosed in co-pending U.S. Ser. No. 08/404,113, and in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn. 179: 116-123, and Ozkaynak, et al. (1992) (JBC, in press), the disclosures of which are incorporated herein by reference. Using the general probing methodology described herein, northern blot hybridizations using probes specific for these morphogens to probe brain, spleen, lung, heart, liver and kidney tissue indicate that kidney-related tissue appears to be the primary expression source for OP-1, with brain, heart and lung tissues being secondary sources. Lung tissue appears to be the primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also are seen in kidney and heart tissue, while the liver appears to be a secondary expression source for BMP5, and the spleen appears to be a secondary expression source for BMP4. GDF-1 appears to be expressed primarily in brain tissue.
Of particular relevance to the present application, OP-1 also is detected in adult rat stomach and gut tissue. Moreover, OP-2 appears to be expressed primarily in early embryonic tissue. Specifically, northern blots of murine embryos and 6-day post-natal animals shows abundant OP2 expression in 8-day embryos. Expression is reduced significantly in 17-day embryos and is not detected in post-natal animals.
In addition, labelled soluble OP-1 (iodinated with 125 I, using standard labelling procedures well known in the art) and injected into the rat tail vein also is localized to the stomach tissue within 30 minutes of injection.
Example 8.
Detecting Morphogenic Protein in Solution by Immunoassay
Morphogens are readily detected in solution with a standard immunoassay, using a polyclonal or monoclonal antibody specific for that protein and standard Western blot, ELISA (enzyme-linked immunoabsorbant assay) or other immunoassay technique well known in the art. A currently preferred, exemplary protocol for an ELISA assay, as well as means for generating morphogen-specific antibody are presented below. Standard protocols for antibody production, Western blot and other immunoassays also are described, for example, in Molecular Cloning A Laboratory Manual, Sambrook et al., eds. 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Standard ELISA technique is described, for example, by Engvall (1980) Methods Enzymol. 70: 419-439.
8.1 Morphogen-Specific Antiserum
Polyclonal antibody was prepared as follows. Each rabbit was given a primary immunization of 100 μg/500 μl E. coli-produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 μl Complete Freund's Adjuvant. The antigen was injected subcutaneously at multiple sites on the back and flanks of the animal. The rabbit was boosted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional boosts and test bleeds were performed at monthly intervals until antibody against OP-1 was detected in the serum using an ELISA assay. Then, the rabbit was boosted monthly with 100 μg of antigen and bled (15 ml per bleed) at days seven and ten after boosting.
8.2 Morphogen-Specific Antibody
Monoclonal antibody specific for a given morphogen was prepared as follows. A mouse was given two injections of E. coli produced OP-1 monomer. The first injection contains 100 μg of OP-1 in complete Freund's adjuvant and was given subcutaneously. The second injection contained 50 μg of OP-1 in incomplete adjuvant and was given intraperitoneally. The mouse then received a total of 230 μg of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period. One week prior to fusion, both mice were boosted intraperitoneally with 100 μg of OP-1 (307-431) and 30 μg of the N-terminal peptide (Ser293-Asn309-Cys) conjugated through the added cysteine to bovine serum albumin with SMCC crosslinking agent. This boost was repeated five days (IP), four days (IP), three days (IP) and one day (IV) prior to fusion. The mouse spleen cells then were fused to commercially available myeloma cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the cell fusion plated and screened for OP-1-specific antibodies using OP-1 (307-431) as antigen. The cell fusion and monoclonal screening then were according to standard procedures well described in standard texts widely available in the art e.g., Maniatis et al. Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press.
8.3 Morphogen ELISA
1 μg/100 μl of affinity-purified polyclonal rabbit IgG specific for OP-1 was added to each well of a 96-well plate and incubated at 37° C. for an hour. The wells were washed four times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimize non-specific binding, the wells are blocked by filling completely with 1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C. The wells are then washed four times with BSB containing 0.1% Tween 20. A 100 μl aliquot of an appropriate dilution of each of the test samples of cell culture supernatant was added to each well in triplicate and incubated at 37° C. for 30 min. After incubation, 100 μl biotinylated rabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) are added to each well and incubated at 37° C. for 30 min. The wells were then washed four times with BSB containing 0.1% Tween 20. 100 μl strepavidin-alkaline (Southern Biotechnology Associates, Inc. Birmingham, Alabama, diluted 1:2000 in BSB containing 0.1% Tween 20 before use) was added to each well and incubated at 37° C. for 30 min. The plates were washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 μl substrate (ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, Md.) was added to each well incubated at room temperature for 15 min. Then, 50 μl amplifier (from the same amplification system kit) is added and incubated for another 15 min at room temperature. The reaction was stopped by the addition of 50 μl 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well was recorded. To quantitate OP-1 in culture media, an OP-1 standard curve was performed in parallel with the test samples.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
__________________________________________________________________________
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#            30                                                           
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#        45                                                               
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#80                                                                       
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- Xaa Xaa Cys Gly Cys Xaa                                                 
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#    60                                                                   
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#80                                                                       
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#                95                                                       
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#           110                                                           
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#       125                                                               
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#   135                                                                   
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#        45                                                               
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#    60                                                                   
- Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Le - #u Asn Ser Tyr Met Asn     
#80                                                                       
- Ala Thr Asn His Ala Ile Val Gln Thr Leu Va - #l His Phe Ile Asn Pro     
#                95                                                       
- Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Th - #r Gln Leu Asn Ala Ile     
#           110                                                           
- Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Va - #l Ile Leu Lys Lys Tyr     
#       125                                                               
- Arg Asn Met Val Val Arg Ala Cys Gly Cys Hi - #s                         
#   135                                                                   
- (2) INFORMATION FOR SEQ ID NO:7:                                        
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 139 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..139                                            
#/note= "HOP-2 (MATURE FORM)"ON:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                             
- Ala Val Arg Pro Leu Arg Arg Arg Gln Pro Ly - #s Lys Ser Asn Glu Leu     
#                15                                                       
- Pro Gln Ala Asn Arg Leu Pro Gly Ile Phe As - #p Asp Val His Gly Ser     
#            30                                                           
- His Gly Arg Gln Val Cys Arg Arg His Glu Le - #u Tyr Val Ser Phe Gln     
#        45                                                               
- Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pr - #o Gln Gly Tyr Ser Ala     
#    60                                                                   
- Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Le - #u Asp Ser Cys Met Asn     
#80                                                                       
- Ala Thr Asn His Ala Ile Leu Gln Ser Leu Va - #l His Leu Met Lys Pro     
#                95                                                       
- Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Th - #r Lys Leu Ser Ala Thr     
#           110                                                           
- Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Va - #l Ile Leu Arg Lys His     
#       125                                                               
- Arg Asn Met Val Val Lys Ala Cys Gly Cys Hi - #s                         
#   135                                                                   
- (2) INFORMATION FOR SEQ ID NO:8:                                        
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 139 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..139                                            
#/note= "MOP-2 (MATURE FORM)"ON:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                             
- Ala Ala Arg Pro Leu Lys Arg Arg Gln Pro Ly - #s Lys Thr Asn Glu Leu     
#                15                                                       
- Pro His Pro Asn Lys Leu Pro Gly Ile Phe As - #p Asp Gly His Gly Ser     
#            30                                                           
- Arg Gly Arg Glu Val Cys Arg Arg His Glu Le - #u Tyr Val Ser Phe Arg     
#        45                                                               
- Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pr - #o Gln Gly Tyr Ser Ala     
#    60                                                                   
- Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Le - #u Asp Ser Cys Met Asn     
#80                                                                       
- Ala Thr Asn His Ala Ile Leu Gln Ser Leu Va - #l His Leu Met Lys Pro     
#                95                                                       
- Asp Val Val Pro Lys Ala Cys Cys Ala Pro Th - #r Lys Leu Ser Ala Thr     
#           110                                                           
- Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Va - #l Ile Leu Arg Lys His     
#       125                                                               
- Arg Asn Met Val Val Lys Ala Cys Gly Cys Hi - #s                         
#   135                                                                   
- (2) INFORMATION FOR SEQ ID NO:9:                                        
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 101 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..101                                            
#/note= "CBMP-2A(FX)"NFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                             
- Cys Lys Arg His Pro Leu Tyr Val Asp Phe Se - #r Asp Val Gly Trp Asn     
#                15                                                       
- Asp Trp Ile Val Ala Pro Pro Gly Tyr His Al - #a Phe Tyr Cys His Gly     
#            30                                                           
- Glu Cys Pro Phe Pro Leu Ala Asp His Leu As - #n Ser Thr Asn His Ala     
#        45                                                               
- Ile Val Gln Thr Leu Val Asn Ser Val Asn Se - #r Lys Ile Pro Lys Ala     
#    60                                                                   
- Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Se - #r Met Leu Tyr Leu Asp     
#80                                                                       
- Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gl - #n Asp Met Val Val Glu     
#                95                                                       
- Gly Cys Gly Cys Arg                                                     
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:10:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 101 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..101                                            
#/note= "CBMP-2B(FX)"NFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                            
- Cys Arg Arg His Ser Leu Tyr Val Asp Phe Se - #r Asp Val Gly Trp Asn     
#                15                                                       
- Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Al - #a Phe Tyr Cys His Gly     
#            30                                                           
- Asp Cys Pro Phe Pro Leu Ala Asp His Leu As - #n Ser Thr Asn His Ala     
#        45                                                               
- Ile Val Gln Thr Leu Val Asn Ser Val Asn Se - #r Ser Ile Pro Lys Ala     
#    60                                                                   
- Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Se - #r Met Leu Tyr Leu Asp     
#80                                                                       
- Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gl - #n Glu Met Val Val Glu     
#                95                                                       
- Gly Cys Gly Cys Arg                                                     
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:11:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/note= "DPP(FX)"ER INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                            
- Cys Arg Arg His Ser Leu Tyr Val Asp Phe Se - #r Asp Val Gly Trp Asp     
#                15                                                       
- Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Al - #a Tyr Tyr Cys His Gly     
#            30                                                           
- Lys Cys Pro Phe Pro Leu Ala Asp His Phe As - #n Ser Thr Asn His Ala     
#        45                                                               
- Val Val Gln Thr Leu Val Asn Asn Asn Asn Pr - #o Gly Lys Val Pro Lys     
#    60                                                                   
- Ala Cys Cys Val Pro Thr Gln Leu Asp Ser Va - #l Ala Met Leu Tyr Leu     
#80                                                                       
- Asn Asp Gln Ser Thr Val Val Leu Lys Asn Ty - #r Gln Glu Met Thr Val     
#                95                                                       
- Val Gly Cys Gly Cys Arg                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:12:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/note= "VGL(FX)"ER INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                            
- Cys Lys Lys Arg His Leu Tyr Val Glu Phe Ly - #s Asp Val Gly Trp Gln     
#                15                                                       
- Asn Trp Val Ile Ala Pro Gln Gly Tyr Met Al - #a Asn Tyr Cys Tyr Gly     
#            30                                                           
- Glu Cys Pro Tyr Pro Leu Thr Glu Ile Leu As - #n Gly Ser Asn His Ala     
#        45                                                               
- Ile Leu Gln Thr Leu Val His Ser Ile Glu Pr - #o Glu Asp Ile Pro Leu     
#    60                                                                   
- Pro Cys Cys Val Pro Thr Lys Met Ser Pro Il - #e Ser Met Leu Phe Tyr     
#80                                                                       
- Asp Asn Asn Asp Asn Val Val Leu Arg His Ty - #r Glu Asn Met Ala Val     
#                95                                                       
- Asp Glu Cys Gly Cys Arg                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:13:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/note= "VGR-1(FX)" INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                            
- Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gl - #n Asp Val Gly Trp Gln     
#                15                                                       
- Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Al - #a Asn Tyr Cys Asp Gly     
#            30                                                           
- Glu Cys Ser Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala     
#        45                                                               
- Ile Val Gln Thr Leu Val His Val Met Asn Pr - #o Glu Tyr Val Pro Lys     
#    60                                                                   
- Pro Cys Cys Ala Pro Thr Lys Val Asn Ala Il - #e Ser Val Leu Tyr Phe     
#80                                                                       
- Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Ty - #r Arg Asn Met Val Val     
#                95                                                       
- Arg Ala Cys Gly Cys His                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:14:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 106 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..106                                            
#/note= "GDF-1 (FX)"INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                            
- Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Ar - #g Glu Val Gly Trp His     
#                15                                                       
- Arg Trp Val Ile Ala Pro Arg Gly Phe Leu Al - #a Asn Tyr Cys Gln Gly     
#            30                                                           
- Gln Cys Ala Leu Pro Val Ala Leu Ser Gly Se - #r Gly Gly Pro Pro Ala     
#        45                                                               
- Leu Asn His Ala Val Leu Arg Ala Leu Met Hi - #s Ala Ala Ala Pro Gly     
#    60                                                                   
- Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Ar - #g Leu Ser Pro Ile Ser     
#80                                                                       
- Val Leu Phe Phe Asp Asn Ser Asp Asn Val Va - #l Leu Arg Gln Tyr Glu     
#                95                                                       
- Asp Met Val Val Asp Glu Cys Gly Cys Arg                                 
#           105                                                           
- (2) INFORMATION FOR SEQ ID NO:15:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 5 amino                                             
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: peptide                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                            
- Cys Xaa Xaa Xaa Xaa                                                     
1               5                                                         
- (2) INFORMATION FOR SEQ ID NO:16:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1822 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 49..1341                                          
#/product= "HOP-1"R INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                            
#CAC GTG      57AGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG                
#                 Met - # His Val                                         
# 1                                                                       
- CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TT - #C GTG GCG CTC TGG GCA     
 105                                                                      
Arg Ser Leu Arg Ala Ala Ala Pro His Ser Ph - #e Val Ala Leu Trp Ala       
#      15                                                                 
- CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GA - #C TTC AGC CTG GAC AAC     
 153                                                                      
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala As - #p Phe Ser Leu Asp Asn       
# 35                                                                      
- GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CT - #C CGC AGC CAG GAG CGG     
 201                                                                      
Glu Val His Ser Ser Phe Ile His Arg Arg Le - #u Arg Ser Gln Glu Arg       
#                 50                                                      
- CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TT - #G GGC TTG CCC CAC CGC     
 249                                                                      
Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Le - #u Gly Leu Pro His Arg       
#             65                                                          
- CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TC - #G GCA CCC ATG TTC ATG     
 297                                                                      
Pro Arg Pro His Leu Gln Gly Lys His Asn Se - #r Ala Pro Met Phe Met       
#         80                                                              
- CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GA - #G GGC GGC GGG CCC GGC     
 345                                                                      
Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Gl - #u Gly Gly Gly Pro Gly       
#     95                                                                  
- GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GT - #C TTC AGT ACC CAG GGC     
 393                                                                      
Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Va - #l Phe Ser Thr Gln Gly       
100                 1 - #05                 1 - #10                 1 -   
#15                                                                       
- CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TT - #C CTC ACC GAC GCC GAC     
 441                                                                      
Pro Pro Leu Ala Ser Leu Gln Asp Ser His Ph - #e Leu Thr Asp Ala Asp       
#               130                                                       
- ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CA - #T GAC AAG GAA TTC TTC     
 489                                                                      
Met Val Met Ser Phe Val Asn Leu Val Glu Hi - #s Asp Lys Glu Phe Phe       
#           145                                                           
- CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TT - #T GAT CTT TCC AAG ATC     
 537                                                                      
His Pro Arg Tyr His His Arg Glu Phe Arg Ph - #e Asp Leu Ser Lys Ile       
#       160                                                               
- CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TT - #C CGG ATC TAC AAG GAC     
 585                                                                      
Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Ph - #e Arg Ile Tyr Lys Asp       
#   175                                                                   
- TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TT - #C CGG ATC AGC GTT TAT     
 633                                                                      
Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Ph - #e Arg Ile Ser Val Tyr       
180                 1 - #85                 1 - #90                 1 -   
#95                                                                       
- CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TC - #G GAT CTC TTC CTG CTC     
 681                                                                      
Gln Val Leu Gln Glu His Leu Gly Arg Glu Se - #r Asp Leu Phe Leu Leu       
#               210                                                       
- GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GG - #C TGG CTG GTG TTT GAC     
 729                                                                      
Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gl - #y Trp Leu Val Phe Asp       
#           225                                                           
- ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AA - #T CCG CGG CAC AAC CTG     
 777                                                                      
Ile Thr Ala Thr Ser Asn His Trp Val Val As - #n Pro Arg His Asn Leu       
#       240                                                               
- GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GG - #G CAG AGC ATC AAC CCC     
 825                                                                      
Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gl - #y Gln Ser Ile Asn Pro       
#   255                                                                   
- AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CC - #C CAG AAC AAG CAG CCC     
 873                                                                      
Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pr - #o Gln Asn Lys Gln Pro       
260                 2 - #65                 2 - #70                 2 -   
#75                                                                       
- TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GT - #C CAC TTC CGC AGC ATC     
 921                                                                      
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Va - #l His Phe Arg Ser Ile       
#               290                                                       
- CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AA - #C CGC TCC AAG ACG CCC     
 969                                                                      
Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln As - #n Arg Ser Lys Thr Pro       
#           305                                                           
- AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GT - #G GCA GAG AAC AGC AGC     
1017                                                                      
Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Va - #l Ala Glu Asn Ser Ser       
#       320                                                               
- AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GA - #G CTG TAT GTC AGC TTC     
1065                                                                      
Ser Asp Gln Arg Gln Ala Cys Lys Lys His Gl - #u Leu Tyr Val Ser Phe       
#   335                                                                   
- CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GC - #G CCT GAA GGC TAC GCC     
1113                                                                      
Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Al - #a Pro Glu Gly Tyr Ala       
340                 3 - #45                 3 - #50                 3 -   
#55                                                                       
- GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CC - #T CTG AAC TCC TAC ATG     
1161                                                                      
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pr - #o Leu Asn Ser Tyr Met       
#               370                                                       
- AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CT - #G GTC CAC TTC ATC AAC     
1209                                                                      
Asn Ala Thr Asn His Ala Ile Val Gln Thr Le - #u Val His Phe Ile Asn       
#           385                                                           
- CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CC - #C ACG CAG CTC AAT GCC     
1257                                                                      
Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pr - #o Thr Gln Leu Asn Ala       
#       400                                                               
- ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AA - #C GTC ATC CTG AAG AAA     
1305                                                                      
Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser As - #n Val Ile Leu Lys Lys       
#   415                                                                   
- TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TG - #C CAC TAGCTCCTCC          
1351                                                                      
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cy - #s His                       
420                 4 - #25                 4 - #30                       
- GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CC - #TTGGCCAG   
1411                                                                      
- GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA AC - #TTTAAAGG   
1471                                                                      
- TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CA - #GTGGCAGC   
1531                                                                      
- ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AA - #AAAACAAC   
1591                                                                      
- GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TG - #CACGGACT   
1651                                                                      
- CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GG - #AGGAAGGG   
1711                                                                      
- GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CC - #GGAAGTTC   
1771                                                                      
#           1822CACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A                
- (2) INFORMATION FOR SEQ ID NO:17:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 431 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                            
- Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala     
#                 15                                                      
- Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser     
#             30                                                          
- Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser     
#         45                                                              
- Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu     
#     60                                                                  
- Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro     
# 80                                                                      
- Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Gly Gly     
#                 95                                                      
- Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Ty - #r Lys Ala Val Phe Ser     
#           110                                                           
- Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln As - #p Ser His Phe Leu Thr     
#       125                                                               
- Asp Ala Asp Met Val Met Ser Phe Val Asn Le - #u Val Glu His Asp Lys     
#   140                                                                   
- Glu Phe Phe His Pro Arg Tyr His His Arg Gl - #u Phe Arg Phe Asp Leu     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Al - #a Ala Glu Phe Arg Ile     
#               175                                                       
- Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp As - #n Glu Thr Phe Arg Ile     
#           190                                                           
- Ser Val Tyr Gln Val Leu Gln Glu His Leu Gl - #y Arg Glu Ser Asp Leu     
#       205                                                               
- Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Se - #r Glu Glu Gly Trp Leu     
#   220                                                                   
- Val Phe Asp Ile Thr Ala Thr Ser Asn His Tr - #p Val Val Asn Pro Arg     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- His Asn Leu Gly Leu Gln Leu Ser Val Glu Th - #r Leu Asp Gly Gln Ser     
#               255                                                       
- Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Ar - #g His Gly Pro Gln Asn     
#           270                                                           
- Lys Gln Pro Phe Met Val Ala Phe Phe Lys Al - #a Thr Glu Val His Phe     
#       285                                                               
- Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Ar - #g Ser Gln Asn Arg Ser     
#   300                                                                   
- Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Me - #t Ala Asn Val Ala Glu     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Ly - #s Lys His Glu Leu Tyr     
#               335                                                       
- Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Tr - #p Ile Ile Ala Pro Glu     
#           350                                                           
- Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asn     
#       365                                                               
- Ser Tyr Met Asn Ala Thr Asn His Ala Ile Va - #l Gln Thr Leu Val His     
#   380                                                                   
- Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cy - #s Cys Ala Pro Thr Gln     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp As - #p Ser Ser Asn Val Ile     
#               415                                                       
- Leu Lys Lys Tyr Arg Asn Met Val Val Arg Al - #a Cys Gly Cys His         
#           430                                                           
- (2) INFORMATION FOR SEQ ID NO:18:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1873 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 104..1393                                         
#/product= "MOP1 (CDNA)"RMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                            
- CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CC - #ACCTGGGG   
  60                                                                      
- CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CA - #C GTG CGC     
 115                                                                      
#            Met His Val Ar - #g                                          
#              1                                                          
- TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GT - #G GCG CTC TGG GCG CCT     
 163                                                                      
Ser Leu Arg Ala Ala Ala Pro His Ser Phe Va - #l Ala Leu Trp Ala Pro       
#  20                                                                     
- CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TT - #C AGC CTG GAC AAC GAG     
 211                                                                      
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Ph - #e Ser Leu Asp Asn Glu       
#                 35                                                      
- GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CG - #C AGC CAG GAG CGG CGG     
 259                                                                      
Val His Ser Ser Phe Ile His Arg Arg Leu Ar - #g Ser Gln Glu Arg Arg       
#             50                                                          
- GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GG - #G TTG CCC CAT CGC CCG     
 307                                                                      
Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gl - #y Leu Pro His Arg Pro       
#         65                                                              
- CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GC - #G CCC ATG TTC ATG TTG     
 355                                                                      
Arg Pro His Leu Gln Gly Lys His Asn Ser Al - #a Pro Met Phe Met Leu       
#     80                                                                  
- GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AG - #C GGG CCG GAC GGA CAG     
 403                                                                      
Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Se - #r Gly Pro Asp Gly Gln       
#100                                                                      
- GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AG - #T ACC CAG GGC CCC CCT     
 451                                                                      
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Se - #r Thr Gln Gly Pro Pro       
#               115                                                       
- TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC AC - #T GAC GCC GAC ATG GTC     
 499                                                                      
Leu Ala Ser Leu Gln Asp Ser His Phe Leu Th - #r Asp Ala Asp Met Val       
#           130                                                           
- ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AA - #A GAA TTC TTC CAC CCT     
 547                                                                      
Met Ser Phe Val Asn Leu Val Glu His Asp Ly - #s Glu Phe Phe His Pro       
#       145                                                               
- CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CT - #T TCC AAG ATC CCC GAG     
 595                                                                      
Arg Tyr His His Arg Glu Phe Arg Phe Asp Le - #u Ser Lys Ile Pro Glu       
#   160                                                                   
- GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG AT - #C TAT AAG GAC TAC ATC     
 643                                                                      
Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Il - #e Tyr Lys Asp Tyr Ile       
165                 1 - #70                 1 - #75                 1 -   
#80                                                                       
- CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG AT - #C ACA GTC TAT CAG GTG     
 691                                                                      
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Il - #e Thr Val Tyr Gln Val       
#               195                                                       
- CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CT - #C TTC TTG CTG GAC AGC     
 739                                                                      
Leu Gln Glu His Ser Gly Arg Glu Ser Asp Le - #u Phe Leu Leu Asp Ser       
#           210                                                           
- CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TT - #G GTG TTT GAT ATC ACA     
 787                                                                      
Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Le - #u Val Phe Asp Ile Thr       
#       225                                                               
- GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CG - #G CAC AAC CTG GGC TTA     
 835                                                                      
Ala Thr Ser Asn His Trp Val Val Asn Pro Ar - #g His Asn Leu Gly Leu       
#   240                                                                   
- CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AG - #C ATC AAC CCC AAG TTG     
 883                                                                      
Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Se - #r Ile Asn Pro Lys Leu       
245                 2 - #50                 2 - #55                 2 -   
#60                                                                       
- GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AA - #C AAG CAA CCC TTC ATG     
 931                                                                      
Ala Gly Leu Ile Gly Arg His Gly Pro Gln As - #n Lys Gln Pro Phe Met       
#               275                                                       
- GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CT - #C CGT AGT ATC CGG TCC     
 979                                                                      
Val Ala Phe Phe Lys Ala Thr Glu Val His Le - #u Arg Ser Ile Arg Ser       
#           290                                                           
- ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TC - #C AAG ACG CCA AAG AAC     
1027                                                                      
Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Se - #r Lys Thr Pro Lys Asn       
#       305                                                               
- CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GA - #A AAC AGC AGC AGT GAC     
1075                                                                      
Gln Glu Ala Leu Arg Met Ala Ser Val Ala Gl - #u Asn Ser Ser Ser Asp       
#   320                                                                   
- CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TA - #C GTC AGC TTC CGA GAC     
1123                                                                      
Gln Arg Gln Ala Cys Lys Lys His Glu Leu Ty - #r Val Ser Phe Arg Asp       
325                 3 - #30                 3 - #35                 3 -   
#40                                                                       
- CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GA - #A GGC TAT GCT GCC TAC     
1171                                                                      
Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Gl - #u Gly Tyr Ala Ala Tyr       
#               355                                                       
- TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AA - #C TCC TAC ATG AAC GCC     
1219                                                                      
Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu As - #n Ser Tyr Met Asn Ala       
#           370                                                           
- ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CA - #C TTC ATC AAC CCA GAC     
1267                                                                      
Thr Asn His Ala Ile Val Gln Thr Leu Val Hi - #s Phe Ile Asn Pro Asp       
#       385                                                               
- ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CA - #G CTC AAC GCC ATC TCT     
1315                                                                      
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gl - #n Leu Asn Ala Ile Ser       
#   400                                                                   
- GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC AT - #C CTG AAG AAG TAC AGA     
1363                                                                      
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Il - #e Leu Lys Lys Tyr Arg       
405                 4 - #10                 4 - #15                 4 -   
#20                                                                       
- AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TA - #GCTCTTCC TGAGACCCTG       
1413                                                                      
Asn Met Val Val Arg Ala Cys Gly Cys His                                   
#               430                                                       
- ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TC - #TCTCACTG   
1473                                                                      
- CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TC - #CCAACCGG   
1533                                                                      
- AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CT - #TTCCTTCT   
1593                                                                      
- GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CA - #GGAAAAAT   
1653                                                                      
- GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TT - #TGAGGAGT   
1713                                                                      
- AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GG - #CGCTGGCG   
1773                                                                      
- TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TA - #AAACCCAT   
1833                                                                      
#  1873            AAAA AAAAAAAAAA AAAAGAATTC                             
- (2) INFORMATION FOR SEQ ID NO:19:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 430 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                            
- Met His Val Arg Ser Leu Arg Ala Ala Ala Pr - #o His Ser Phe Val Ala     
#                 15                                                      
- Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Al - #a Leu Ala Asp Phe Ser     
#             30                                                          
- Leu Asp Asn Glu Val His Ser Ser Phe Ile Hi - #s Arg Arg Leu Arg Ser     
#         45                                                              
- Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Le - #u Ser Ile Leu Gly Leu     
#     60                                                                  
- Pro His Arg Pro Arg Pro His Leu Gln Gly Ly - #s His Asn Ser Ala Pro     
# 80                                                                      
- Met Phe Met Leu Asp Leu Tyr Asn Ala Met Al - #a Val Glu Glu Ser Gly     
#                 95                                                      
- Pro Asp Gly Gln Gly Phe Ser Tyr Pro Tyr Ly - #s Ala Val Phe Ser Thr     
#           110                                                           
- Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Se - #r His Phe Leu Thr Asp     
#       125                                                               
- Ala Asp Met Val Met Ser Phe Val Asn Leu Va - #l Glu His Asp Lys Glu     
#   140                                                                   
- Phe Phe His Pro Arg Tyr His His Arg Glu Ph - #e Arg Phe Asp Leu Ser     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Al - #a Glu Phe Arg Ile Tyr     
#               175                                                       
- Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Gl - #u Thr Phe Gln Ile Thr     
#           190                                                           
- Val Tyr Gln Val Leu Gln Glu His Ser Gly Ar - #g Glu Ser Asp Leu Phe     
#       205                                                               
- Leu Leu Asp Ser Arg Thr Ile Trp Ala Ser Gl - #u Glu Gly Trp Leu Val     
#   220                                                                   
- Phe Asp Ile Thr Ala Thr Ser Asn His Trp Va - #l Val Asn Pro Arg His     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Le - #u Asp Gly Gln Ser Ile     
#               255                                                       
- Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg Hi - #s Gly Pro Gln Asn Lys     
#           270                                                           
- Gln Pro Phe Met Val Ala Phe Phe Lys Ala Th - #r Glu Val His Leu Arg     
#       285                                                               
- Ser Ile Arg Ser Thr Gly Gly Lys Gln Arg Se - #r Gln Asn Arg Ser Lys     
#   300                                                                   
- Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Al - #a Ser Val Ala Glu Asn     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Ly - #s His Glu Leu Tyr Val     
#               335                                                       
- Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Il - #e Ile Ala Pro Glu Gly     
#           350                                                           
- Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Al - #a Phe Pro Leu Asn Ser     
#       365                                                               
- Tyr Met Asn Ala Thr Asn His Ala Ile Val Gl - #n Thr Leu Val His Phe     
#   380                                                                   
- Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cy - #s Ala Pro Thr Gln Leu     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Se - #r Ser Asn Val Ile Leu     
#               415                                                       
- Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cy - #s Gly Cys His             
#           430                                                           
- (2) INFORMATION FOR SEQ ID NO:20:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1723 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 490..1695                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                            
- GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TG - #GCACGGCA   
  60                                                                      
- GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GC - #AACAGCTC   
 120                                                                      
- CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TG - #CTCGGACC   
 180                                                                      
- GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GA - #GTCCCAGT   
 240                                                                      
- CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CA - #GGAGCCAG   
 300                                                                      
- GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CC - #GCCCGTCC   
 360                                                                      
- CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GG - #GCGTCCCC   
 420                                                                      
- AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TG - #AGCGCCCC   
 480                                                                      
- CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG - # CTC CTG GGC CTG       
 528                                                                      
#Gly Pro Leu Trp Leu Leu Gly Leu                                          
#         10                                                              
- GCG CTA TGC GCG CTG GGC GGG GGC GGC CCC GG - #C CTG CGA CCC CCG CCC     
 576                                                                      
Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gl - #y Leu Arg Pro Pro Pro       
#     25                                                                  
- GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GA - #G CGC CGG GAC GTG CAG     
 624                                                                      
Gly Cys Pro Gln Arg Arg Leu Gly Ala Arg Gl - #u Arg Arg Asp Val Gln       
# 45                                                                      
- CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GG - #G CGG CCC CGG CCC CGC     
 672                                                                      
Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gl - #y Arg Pro Arg Pro Arg       
#                 60                                                      
- GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TC - #C GCG CCG CTC TTC ATG     
 720                                                                      
Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Se - #r Ala Pro Leu Phe Met       
#             75                                                          
- CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GA - #C GAC GAG GAC GGC GCG     
 768                                                                      
Leu Asp Leu Tyr His Ala Met Ala Gly Asp As - #p Asp Glu Asp Gly Ala       
#         90                                                              
- CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CT - #G GTC ATG AGC TTC GTT     
 816                                                                      
Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Le - #u Val Met Ser Phe Val       
#    105                                                                  
- AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CA - #C CAG GAG CCC CAT TGG     
 864                                                                      
Asn Met Val Glu Arg Asp Arg Ala Leu Gly Hi - #s Gln Glu Pro His Trp       
110                 1 - #15                 1 - #20                 1 -   
#25                                                                       
- AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CC - #G GCT GGG GAG GCG GTC     
 912                                                                      
Lys Glu Phe Arg Phe Asp Leu Thr Gln Ile Pr - #o Ala Gly Glu Ala Val       
#               140                                                       
- ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CC - #C AGC ATC CAC CTG CTC     
 960                                                                      
Thr Ala Ala Glu Phe Arg Ile Tyr Lys Val Pr - #o Ser Ile His Leu Leu       
#           155                                                           
- AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GT - #G GTC CAG GAG CAG TCC     
1008                                                                      
Asn Arg Thr Leu His Val Ser Met Phe Gln Va - #l Val Gln Glu Gln Ser       
#       170                                                               
- AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CT - #T CAG ACG CTC CGA GCT     
1056                                                                      
Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Le - #u Gln Thr Leu Arg Ala       
#   185                                                                   
- GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC AC - #A GCA GCC AGT GAC TGC     
1104                                                                      
Gly Asp Glu Gly Trp Leu Val Leu Asp Val Th - #r Ala Ala Ser Asp Cys       
190                 1 - #95                 2 - #00                 2 -   
#05                                                                       
- TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CT - #C CGC CTC TAT GTG GAG     
1152                                                                      
Trp Leu Leu Lys Arg His Lys Asp Leu Gly Le - #u Arg Leu Tyr Val Glu       
#               220                                                       
- ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CT - #G GCC GGC CTG CTG GGT     
1200                                                                      
Thr Glu Asp Gly His Ser Val Asp Pro Gly Le - #u Ala Gly Leu Leu Gly       
#           235                                                           
- CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GT - #G GTC ACT TTC TTC AGG     
1248                                                                      
Gln Arg Ala Pro Arg Ser Gln Gln Pro Phe Va - #l Val Thr Phe Phe Arg       
#       250                                                               
- GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GC - #A GTG AGG CCA CTG AGG     
1296                                                                      
Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Al - #a Val Arg Pro Leu Arg       
#   265                                                                   
- AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CC - #G CAG GCC AAC CGA CTC     
1344                                                                      
Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pr - #o Gln Ala Asn Arg Leu       
270                 2 - #75                 2 - #80                 2 -   
#85                                                                       
- CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CA - #C GGC CGG CAG GTC TGC     
1392                                                                      
Pro Gly Ile Phe Asp Asp Val His Gly Ser Hi - #s Gly Arg Gln Val Cys       
#               300                                                       
- CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GA - #C CTC GGC TGG CTG GAC     
1440                                                                      
Arg Arg His Glu Leu Tyr Val Ser Phe Gln As - #p Leu Gly Trp Leu Asp       
#           315                                                           
- TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TA - #T TAC TGT GAG GGG GAG     
1488                                                                      
Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Ty - #r Tyr Cys Glu Gly Glu       
#       330                                                               
- TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GC - #C ACC AAC CAC GCC ATC     
1536                                                                      
Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Al - #a Thr Asn His Ala Ile       
#   345                                                                   
- CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AA - #C GCA GTC CCC AAG GCG     
1584                                                                      
Leu Gln Ser Leu Val His Leu Met Lys Pro As - #n Ala Val Pro Lys Ala       
350                 3 - #55                 3 - #60                 3 -   
#65                                                                       
- TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TC - #T GTG CTC TAC TAT GAC     
1632                                                                      
Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Se - #r Val Leu Tyr Tyr Asp       
#               380                                                       
- AGC AGC AAC AAC GTC ATC CTG CGC AAA CAC CG - #C AAC ATG GTG GTC AAG     
1680                                                                      
Ser Ser Asn Asn Val Ile Leu Arg Lys His Ar - #g Asn Met Val Val Lys       
#           395                                                           
#                 172 - #3TCAGCC CGCCCAGCCC TACTGCAG                      
Ala Cys Gly Cys His                                                       
        400                                                               
- (2) INFORMATION FOR SEQ ID NO:21:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 402 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                            
- Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Le - #u Gly Leu Ala Leu Cys     
#                 15                                                      
- Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pr - #o Pro Pro Gly Cys Pro     
#             30                                                          
- Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg As - #p Val Gln Arg Glu Ile     
#         45                                                              
- Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Ar - #g Pro Arg Ala Pro Pro     
#     60                                                                  
- Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Le - #u Phe Met Leu Asp Leu     
# 80                                                                      
- Tyr His Ala Met Ala Gly Asp Asp Asp Glu As - #p Gly Ala Pro Ala Glu     
#                 95                                                      
- Arg Arg Leu Gly Arg Ala Asp Leu Val Met Se - #r Phe Val Asn Met Val     
#           110                                                           
- Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pr - #o His Trp Lys Glu Phe     
#       125                                                               
- Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Gl - #u Ala Val Thr Ala Ala     
#   140                                                                   
- Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile Hi - #s Leu Leu Asn Arg Thr     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Leu His Val Ser Met Phe Gln Val Val Gln Gl - #u Gln Ser Asn Arg Glu     
#               175                                                       
- Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Le - #u Arg Ala Gly Asp Glu     
#           190                                                           
- Gly Trp Leu Val Leu Asp Val Thr Ala Ala Se - #r Asp Cys Trp Leu Leu     
#       205                                                               
- Lys Arg His Lys Asp Leu Gly Leu Arg Leu Ty - #r Val Glu Thr Glu Asp     
#   220                                                                   
- Gly His Ser Val Asp Pro Gly Leu Ala Gly Le - #u Leu Gly Gln Arg Ala     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Pro Arg Ser Gln Gln Pro Phe Val Val Thr Ph - #e Phe Arg Ala Ser Pro     
#               255                                                       
- Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pr - #o Leu Arg Arg Arg Gln     
#           270                                                           
- Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala As - #n Arg Leu Pro Gly Ile     
#       285                                                               
- Phe Asp Asp Val His Gly Ser His Gly Arg Gl - #n Val Cys Arg Arg His     
#   300                                                                   
- Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Tr - #p Leu Asp Trp Val Ile     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Gl - #u Gly Glu Cys Ser Phe     
#               335                                                       
- Pro Leu Asp Ser Cys Met Asn Ala Thr Asn Hi - #s Ala Ile Leu Gln Ser     
#           350                                                           
- Leu Val His Leu Met Lys Pro Asn Ala Val Pr - #o Lys Ala Cys Cys Ala     
#       365                                                               
- Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Ty - #r Tyr Asp Ser Ser Asn     
#   380                                                                   
- Asn Val Ile Leu Arg Lys His Arg Asn Met Va - #l Val Lys Ala Cys Gly     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Cys His                                                                 
- (2) INFORMATION FOR SEQ ID NO:22:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1926 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 93..1289                                          
#/product= "MOP2 CDNA"FORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                            
- GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CC - #GACCAGCT   
  60                                                                      
- ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT - # CCC GGG CCA     
 113                                                                      
#Met Ala Met Arg Pro Gly Pro                                              
#  1               5                                                      
- CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CT - #G GGA GGC GGC CAC GGT     
 161                                                                      
Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Le - #u Gly Gly Gly His Gly       
#         20                                                              
- CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CG - #C CTG GGA GCG CGC GAG     
 209                                                                      
Pro Arg Pro Pro His Thr Cys Pro Gln Arg Ar - #g Leu Gly Ala Arg Glu       
#     35                                                                  
- CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GT - #G CTC GGG CTA CCG GGA     
 257                                                                      
Arg Arg Asp Met Gln Arg Glu Ile Leu Ala Va - #l Leu Gly Leu Pro Gly       
# 55                                                                      
- CGG CCC CGA CCC CGT GCA CAA CCC GCC GCT GC - #C CGG CAG CCA GCG TCC     
 305                                                                      
Arg Pro Arg Pro Arg Ala Gln Pro Ala Ala Al - #a Arg Gln Pro Ala Ser       
#                 70                                                      
- GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GC - #C ATG ACC GAT GAC GAC     
 353                                                                      
Ala Pro Leu Phe Met Leu Asp Leu Tyr His Al - #a Met Thr Asp Asp Asp       
#             85                                                          
- GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CG - #T GCC GAC CTG GTC ATG     
 401                                                                      
Asp Gly Gly Pro Pro Gln Ala His Leu Gly Ar - #g Ala Asp Leu Val Met       
#        100                                                              
- AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT AC - #C CTG GGC TAC CAG GAG     
 449                                                                      
Ser Phe Val Asn Met Val Glu Arg Asp Arg Th - #r Leu Gly Tyr Gln Glu       
#   115                                                                   
- CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA AC - #C CAG ATC CCT GCT GGG     
 497                                                                      
Pro His Trp Lys Glu Phe His Phe Asp Leu Th - #r Gln Ile Pro Ala Gly       
120                 1 - #25                 1 - #30                 1 -   
#35                                                                       
- GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TA - #C AAA GAA CCC AGC ACC     
 545                                                                      
Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Ty - #r Lys Glu Pro Ser Thr       
#               150                                                       
- CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC AT - #G TTC GAA GTG GTC CAA     
 593                                                                      
His Pro Leu Asn Thr Thr Leu His Ile Ser Me - #t Phe Glu Val Val Gln       
#           165                                                           
- GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TT - #T TTG GAT CTT CAG ACG     
 641                                                                      
Glu His Ser Asn Arg Glu Ser Asp Leu Phe Ph - #e Leu Asp Leu Gln Thr       
#       180                                                               
- CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CT - #G GAC ATC ACA GCA GCC     
 689                                                                      
Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Le - #u Asp Ile Thr Ala Ala       
#   195                                                                   
- AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GA - #C CTG GGA CTC CGC CTC     
 737                                                                      
Ser Asp Arg Trp Leu Leu Asn His His Lys As - #p Leu Gly Leu Arg Leu       
200                 2 - #05                 2 - #10                 2 -   
#15                                                                       
- TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GA - #T CCT GGC CTG GCT GGT     
 785                                                                      
Tyr Val Glu Thr Ala Asp Gly His Ser Met As - #p Pro Gly Leu Ala Gly       
#               230                                                       
- CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CA - #G CCT TTC ATG GTA ACC     
 833                                                                      
Leu Leu Gly Arg Gln Ala Pro Arg Ser Arg Gl - #n Pro Phe Met Val Thr       
#           245                                                           
- TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GC - #C CCT CGG GCA GCG AGA     
 881                                                                      
Phe Phe Arg Ala Ser Gln Ser Pro Val Arg Al - #a Pro Arg Ala Ala Arg       
#       260                                                               
- CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AA - #C GAG CTT CCG CAC CCC     
 929                                                                      
Pro Leu Lys Arg Arg Gln Pro Lys Lys Thr As - #n Glu Leu Pro His Pro       
#   275                                                                   
- AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CA - #C GGT TCC CGC GGC AGA     
 977                                                                      
Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly Hi - #s Gly Ser Arg Gly Arg       
280                 2 - #85                 2 - #90                 2 -   
#95                                                                       
- GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AG - #C TTC CGT GAC CTT GGC     
1025                                                                      
Glu Val Cys Arg Arg His Glu Leu Tyr Val Se - #r Phe Arg Asp Leu Gly       
#               310                                                       
- TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TA - #C TCT GCC TAT TAC TGT     
1073                                                                      
Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Ty - #r Ser Ala Tyr Tyr Cys       
#           325                                                           
- GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TG - #T ATG AAC GCC ACC AAC     
1121                                                                      
Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cy - #s Met Asn Ala Thr Asn       
#       340                                                               
- CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG AT - #G AAG CCA GAT GTT GTC     
1169                                                                      
His Ala Ile Leu Gln Ser Leu Val His Leu Me - #t Lys Pro Asp Val Val       
#   355                                                                   
- CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AG - #T GCC ACC TCT GTG CTG     
1217                                                                      
Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Se - #r Ala Thr Ser Val Leu       
360                 3 - #65                 3 - #70                 3 -   
#75                                                                       
- TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CG - #T AAA CAC CGT AAC ATG     
1265                                                                      
Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Ar - #g Lys His Arg Asn Met       
#               390                                                       
- GTG GTC AAG GCC TGT GGC TGC CAC TGAGGCCCCG CC - #CAGCATCC TGCTTCTACT    
1319                                                                      
Val Val Lys Ala Cys Gly Cys His                                           
            395                                                           
- ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT TA - #TCATAGCT   
1379                                                                      
- CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA AA - #ATTCTGGT   
1439                                                                      
- CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CT - #CTCCATCC   
1499                                                                      
- TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA AC - #TGAGAGGT   
1559                                                                      
- CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC CT - #CAGCCCAC   
1619                                                                      
- AATGGCAAAT TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT GA - #TCTGGGCT   
1679                                                                      
- CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CA - #TACACTTA   
1739                                                                      
- GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT TGTTAGAAAA AG - #AATCAGAG   
1799                                                                      
- CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC AG - #GAGAATCT   
1859                                                                      
- CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA AA - #AAAAAAAC   
1919                                                                      
#        1926                                                             
- (2) INFORMATION FOR SEQ ID NO:23:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 399 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                            
- Met Ala Met Arg Pro Gly Pro Leu Trp Leu Le - #u Gly Leu Ala Leu Cys     
#                 15                                                      
- Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pr - #o His Thr Cys Pro Gln     
#             30                                                          
- Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Me - #t Gln Arg Glu Ile Leu     
#         45                                                              
- Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pr - #o Arg Ala Gln Pro Ala     
#     60                                                                  
- Ala Ala Arg Gln Pro Ala Ser Ala Pro Leu Ph - #e Met Leu Asp Leu Tyr     
# 80                                                                      
- His Ala Met Thr Asp Asp Asp Asp Gly Gly Pr - #o Pro Gln Ala His Leu     
#                 95                                                      
- Gly Arg Ala Asp Leu Val Met Ser Phe Val As - #n Met Val Glu Arg Asp     
#           110                                                           
- Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Ly - #s Glu Phe His Phe Asp     
#       125                                                               
- Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Th - #r Ala Ala Glu Phe Arg     
#   140                                                                   
- Ile Tyr Lys Glu Pro Ser Thr His Pro Leu As - #n Thr Thr Leu His Ile     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Ser Met Phe Glu Val Val Gln Glu His Ser As - #n Arg Glu Ser Asp Leu     
#               175                                                       
- Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gl - #y Asp Glu Gly Trp Leu     
#           190                                                           
- Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Tr - #p Leu Leu Asn His His     
#       205                                                               
- Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Th - #r Ala Asp Gly His Ser     
#   220                                                                   
- Met Asp Pro Gly Leu Ala Gly Leu Leu Gly Ar - #g Gln Ala Pro Arg Ser     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Arg Gln Pro Phe Met Val Thr Phe Phe Arg Al - #a Ser Gln Ser Pro Val     
#               255                                                       
- Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Ar - #g Arg Gln Pro Lys Lys     
#           270                                                           
- Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pr - #o Gly Ile Phe Asp Asp     
#       285                                                               
- Gly His Gly Ser Arg Gly Arg Glu Val Cys Ar - #g Arg His Glu Leu Tyr     
#   300                                                                   
- Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Tr - #p Val Ile Ala Pro Gln     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cy - #s Ala Phe Pro Leu Asp     
#               335                                                       
- Ser Cys Met Asn Ala Thr Asn His Ala Ile Le - #u Gln Ser Leu Val His     
#           350                                                           
- Leu Met Lys Pro Asp Val Val Pro Lys Ala Cy - #s Cys Ala Pro Thr Lys     
#       365                                                               
- Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Se - #r Ser Asn Asn Val Ile     
#   380                                                                   
- Leu Arg Lys His Arg Asn Met Val Val Lys Al - #a Cys Gly Cys His         
385                 3 - #90                 3 - #95                       
- (2) INFORMATION FOR SEQ ID NO:24:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1368 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 1..1365                                           
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                            
- ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GT - #T GCA GTG CTC GCC TCC     
  48                                                                      
Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Va - #l Ala Val Leu Ala Ser       
#                 15                                                      
- CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GT - #G GCG ACC ACG CCG CCG     
  96                                                                      
Leu Gly Leu Gly Met Val Leu Leu Met Phe Va - #l Ala Thr Thr Pro Pro       
#             30                                                          
- GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC AT - #A GAC AAC GGC AAG GAC     
 144                                                                      
Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Il - #e Asp Asn Gly Lys Asp       
#         45                                                              
- CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GA - #C GAC AAG CTG GAC GTC     
 192                                                                      
Gln Thr Ile Met His Arg Val Leu Ser Glu As - #p Asp Lys Leu Asp Val       
#     60                                                                  
- TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GC - #C GAA CGG CCG ACG CAC     
 240                                                                      
Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile Al - #a Glu Arg Pro Thr His       
# 80                                                                      
- CTG AGC AGC CAC CAG TTG TCG CTG AGG AAG TC - #G GCT CCC AAG TTC CTG     
 288                                                                      
Leu Ser Ser His Gln Leu Ser Leu Arg Lys Se - #r Ala Pro Lys Phe Leu       
#                 95                                                      
- CTG GAC GTC TAC CAC CGC ATC ACG GCG GAG GA - #G GGT CTC AGC GAT CAG     
 336                                                                      
Leu Asp Val Tyr His Arg Ile Thr Ala Glu Gl - #u Gly Leu Ser Asp Gln       
#           110                                                           
- GAT GAG GAC GAC GAC TAC GAA CGC GGC CAT CG - #G TCC AGG AGG AGC GCC     
 384                                                                      
Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Ar - #g Ser Arg Arg Ser Ala       
#       125                                                               
- GAC CTC GAG GAG GAT GAG GGC GAG CAG CAG AA - #G AAC TTC ATC ACC GAC     
 432                                                                      
Asp Leu Glu Glu Asp Glu Gly Glu Gln Gln Ly - #s Asn Phe Ile Thr Asp       
#   140                                                                   
- CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC AT - #C ATC ATG ACC TTC CTG     
 480                                                                      
Leu Asp Lys Arg Ala Ile Asp Glu Ser Asp Il - #e Ile Met Thr Phe Leu       
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- AAC AAG CGC CAC CAC AAT GTG GAC GAA CTG CG - #T CAC GAG CAC GGC CGT     
 528                                                                      
Asn Lys Arg His His Asn Val Asp Glu Leu Ar - #g His Glu His Gly Arg       
#               175                                                       
- CGC CTG TGG TTC GAC GTC TCC AAC GTG CCC AA - #C GAC AAC TAC CTG GTG     
 576                                                                      
Arg Leu Trp Phe Asp Val Ser Asn Val Pro As - #n Asp Asn Tyr Leu Val       
#           190                                                           
- ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AA - #C GAG GGC AAG TGG CTG     
 624                                                                      
Met Ala Glu Leu Arg Ile Tyr Gln Asn Ala As - #n Glu Gly Lys Trp Leu       
#       205                                                               
- ACC GCC AAC AGG GAG TTC ACC ATC ACG GTA TA - #C GCC ATT GGC ACC GGC     
 672                                                                      
Thr Ala Asn Arg Glu Phe Thr Ile Thr Val Ty - #r Ala Ile Gly Thr Gly       
#   220                                                                   
- ACG CTG GGC CAG CAC ACC ATG GAG CCG CTG TC - #C TCG GTG AAC ACC ACC     
 720                                                                      
Thr Leu Gly Gln His Thr Met Glu Pro Leu Se - #r Ser Val Asn Thr Thr       
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- GGG GAC TAC GTG GGC TGG TTG GAG CTC AAC GT - #G ACC GAG GGC CTG CAC     
 768                                                                      
Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Va - #l Thr Glu Gly Leu His       
#               255                                                       
- GAG TGG CTG GTC AAG TCG AAG GAC AAT CAT GG - #C ATC TAC ATT GGA GCA     
 816                                                                      
Glu Trp Leu Val Lys Ser Lys Asp Asn His Gl - #y Ile Tyr Ile Gly Ala       
#           270                                                           
- CAC GCT GTC AAC CGA CCC GAC CGC GAG GTG AA - #G CTG GAC GAC ATT GGA     
 864                                                                      
His Ala Val Asn Arg Pro Asp Arg Glu Val Ly - #s Leu Asp Asp Ile Gly       
#       285                                                               
- CTG ATC CAC CGC AAG GTG GAC GAC GAG TTC CA - #G CCC TTC ATG ATC GGC     
 912                                                                      
Leu Ile His Arg Lys Val Asp Asp Glu Phe Gl - #n Pro Phe Met Ile Gly       
#   300                                                                   
- TTC TTC CGC GGA CCG GAG CTG ATC AAG GCG AC - #G GCC CAC AGC AGC CAC     
 960                                                                      
Phe Phe Arg Gly Pro Glu Leu Ile Lys Ala Th - #r Ala His Ser Ser His       
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA CG - #C AAG CGC AAG AAG TCG     
1008                                                                      
His Arg Ser Lys Arg Ser Ala Ser His Pro Ar - #g Lys Arg Lys Lys Ser       
#               335                                                       
- GTG TCG CCC AAC AAC GTG CCG CTG CTG GAA CC - #G ATG GAG AGC ACG CGC     
1056                                                                      
Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pr - #o Met Glu Ser Thr Arg       
#           350                                                           
- AGC TGC CAG ATG CAG ACC CTG TAC ATA GAC TT - #C AAG GAT CTG GGC TGG     
1104                                                                      
Ser Cys Gln Met Gln Thr Leu Tyr Ile Asp Ph - #e Lys Asp Leu Gly Trp       
#       365                                                               
- CAT GAC TGG ATC ATC GCA CCA GAG GGC TAT GG - #C GCC TTC TAC TGC AGC     
1152                                                                      
His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gl - #y Ala Phe Tyr Cys Ser       
#   380                                                                   
- GGC GAG TGC AAT TTC CCG CTC AAT GCG CAC AT - #G AAC GCC ACG AAC CAT     
1200                                                                      
Gly Glu Cys Asn Phe Pro Leu Asn Ala His Me - #t Asn Ala Thr Asn His       
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- GCG ATC GTC CAG ACC CTG GTC CAC CTG CTG GA - #G CCC AAG AAG GTG CCC     
1248                                                                      
Ala Ile Val Gln Thr Leu Val His Leu Leu Gl - #u Pro Lys Lys Val Pro       
#               415                                                       
- AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GC - #A CTA CCC GTT CTG TAC     
1296                                                                      
Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Al - #a Leu Pro Val Leu Tyr       
#           430                                                           
- CAC CTG AAC GAC GAG AAT GTG AAC CTG AAA AA - #G TAT AGA AAC ATG ATT     
1344                                                                      
His Leu Asn Asp Glu Asn Val Asn Leu Lys Ly - #s Tyr Arg Asn Met Ile       
#       445                                                               
#              1368GG TGC CAT TGA                                         
Val Lys Ser Cys Gly Cys His                                               
#   455                                                                   
- (2) INFORMATION FOR SEQ ID NO:25:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 455 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                            
- Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Va - #l Ala Val Leu Ala Ser     
#                 15                                                      
- Leu Gly Leu Gly Met Val Leu Leu Met Phe Va - #l Ala Thr Thr Pro Pro     
#             30                                                          
- Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Il - #e Asp Asn Gly Lys Asp     
#         45                                                              
- Gln Thr Ile Met His Arg Val Leu Ser Glu As - #p Asp Lys Leu Asp Val     
#     60                                                                  
- Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile Al - #a Glu Arg Pro Thr His     
# 80                                                                      
- Leu Ser Ser His Gln Leu Ser Leu Arg Lys Se - #r Ala Pro Lys Phe Leu     
#                 95                                                      
- Leu Asp Val Tyr His Arg Ile Thr Ala Glu Gl - #u Gly Leu Ser Asp Gln     
#           110                                                           
- Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Ar - #g Ser Arg Arg Ser Ala     
#       125                                                               
- Asp Leu Glu Glu Asp Glu Gly Glu Gln Gln Ly - #s Asn Phe Ile Thr Asp     
#   140                                                                   
- Leu Asp Lys Arg Ala Ile Asp Glu Ser Asp Il - #e Ile Met Thr Phe Leu     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Asn Lys Arg His His Asn Val Asp Glu Leu Ar - #g His Glu His Gly Arg     
#               175                                                       
- Arg Leu Trp Phe Asp Val Ser Asn Val Pro As - #n Asp Asn Tyr Leu Val     
#           190                                                           
- Met Ala Glu Leu Arg Ile Tyr Gln Asn Ala As - #n Glu Gly Lys Trp Leu     
#       205                                                               
- Thr Ala Asn Arg Glu Phe Thr Ile Thr Val Ty - #r Ala Ile Gly Thr Gly     
#   220                                                                   
- Thr Leu Gly Gln His Thr Met Glu Pro Leu Se - #r Ser Val Asn Thr Thr     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Va - #l Thr Glu Gly Leu His     
#               255                                                       
- Glu Trp Leu Val Lys Ser Lys Asp Asn His Gl - #y Ile Tyr Ile Gly Ala     
#           270                                                           
- His Ala Val Asn Arg Pro Asp Arg Glu Val Ly - #s Leu Asp Asp Ile Gly     
#       285                                                               
- Leu Ile His Arg Lys Val Asp Asp Glu Phe Gl - #n Pro Phe Met Ile Gly     
#   300                                                                   
- Phe Phe Arg Gly Pro Glu Leu Ile Lys Ala Th - #r Ala His Ser Ser His     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- His Arg Ser Lys Arg Ser Ala Ser His Pro Ar - #g Lys Arg Lys Lys Ser     
#               335                                                       
- Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pr - #o Met Glu Ser Thr Arg     
#           350                                                           
- Ser Cys Gln Met Gln Thr Leu Tyr Ile Asp Ph - #e Lys Asp Leu Gly Trp     
#       365                                                               
- His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gl - #y Ala Phe Tyr Cys Ser     
#   380                                                                   
- Gly Glu Cys Asn Phe Pro Leu Asn Ala His Me - #t Asn Ala Thr Asn His     
385                 3 - #90                 3 - #95                 4 -   
#00                                                                       
- Ala Ile Val Gln Thr Leu Val His Leu Leu Gl - #u Pro Lys Lys Val Pro     
#               415                                                       
- Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Al - #a Leu Pro Val Leu Tyr     
#           430                                                           
- His Leu Asn Asp Glu Asn Val Asn Leu Lys Ly - #s Tyr Arg Asn Met Ile     
#       445                                                               
- Val Lys Ser Cys Gly Cys His                                             
#   455                                                                   
- (2) INFORMATION FOR SEQ ID NO:26:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 104 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..104                                            
#/label= BMP3 OTHER INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                            
- Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Al - #a Asp Ile Gly Trp Ser     
#                15                                                       
- Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Al - #a Tyr Tyr Cys Ser Gly     
#            30                                                           
- Ala Cys Gln Phe Pro Met Pro Lys Ser Leu Ly - #s Pro Ser Asn His Ala     
#        45                                                               
- Thr Ile Gln Ser Ile Val Ala Arg Ala Val Gl - #y Val Val Pro Gly Ile     
#    60                                                                   
- Pro Glu Pro Cys Cys Val Pro Glu Lys Met Se - #r Ser Leu Ser Ile Leu     
#80                                                                       
- Phe Phe Asp Glu Asn Lys Asn Val Val Leu Ly - #s Val Tyr Pro Asn Met     
#                95                                                       
- Thr Val Glu Ser Cys Ala Cys Arg                                         
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:27:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/label= BMP5 OTHER INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                            
- Cys Lys Lys His Glu Leu Tyr Val Ser Phe Ar - #g Asp Leu Gly Trp Gln     
#                15                                                       
- Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Al - #a Phe Tyr Cys Asp Gly     
#            30                                                           
- Glu Cys Ser Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala     
#        45                                                               
- Ile Val Gln Thr Leu Val His Leu Met Phe Pr - #o Asp His Val Pro Lys     
#    60                                                                   
- Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Il - #e Ser Val Leu Tyr Phe     
#80                                                                       
- Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Ty - #r Arg Asn Met Val Val     
#                95                                                       
- Arg Ser Cys Gly Cys His                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:28:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/label= BMP6 OTHER INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                            
- Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gl - #n Asp Leu Gly Trp Gln     
#                15                                                       
- Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Al - #a Asn Tyr Cys Asp Gly     
#            30                                                           
- Glu Cys Ser Phe Pro Leu Asn Ala His Met As - #n Ala Thr Asn His Ala     
#        45                                                               
- Ile Val Gln Thr Leu Val His Leu Met Asn Pr - #o Glu Tyr Val Pro Lys     
#    60                                                                   
- Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Il - #e Ser Val Leu Tyr Phe     
#80                                                                       
- Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Ty - #r Arg Trp Met Val Val     
#                95                                                       
- Arg Ala Cys Gly Cys His                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:29:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/label= OPX) OTHER INFORMATION:                                          
#"WHEREIN XAA AT EACH POS'N IS INDEPENDENTLY                              
#FROM THE RESIDUES OCCURING AT THE CORRESPONDING                          
#IN THE C-TERMINAL SEQUENCE OF MOUSE OR HUMAN OP - #1 OR                  
               OP2 (SEQ. - # ID NOS. 5,6,7&8 OR 16,18, 20&22"             
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                            
- Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xa - #a Asp Leu Gly Trp Xaa     
#                15                                                       
- Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Al - #a Tyr Tyr Cys Glu Gly     
#            30                                                           
- Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met As - #n Ala Thr Asn His Ala     
#        45                                                               
- Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pr - #o Xaa Xaa Val Pro Lys     
#    60                                                                   
- Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xa - #a Ser Val Leu Tyr Xaa     
#80                                                                       
- Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xa - #a Arg Asn Met Val Val     
#                95                                                       
- Xaa Ala Cys Gly Cys His                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:30:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 97 amino                                            
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..97                                             
#/label= GENERIC-SEQ-5FORMATION:                                          
#AWHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM                         
#ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN                          
               THE SPECI - #FICATION"                                     
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                            
- Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xa - #a Xaa Trp Xaa Xaa Xaa     
#                15                                                       
- Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gl - #y Xaa Cys Xaa Xaa Pro     
#            30                                                           
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Al - #a Xaa Xaa Xaa Xaa Xaa     
#        45                                                               
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Cys Cys Xaa Pro     
#    60                                                                   
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa     
#80                                                                       
- Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Va - #l Xaa Xaa Cys Xaa Cys     
#                95                                                       
- Xaa                                                                     
- (2) INFORMATION FOR SEQ ID NO:31:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 102 amino                                           
          (B) TYPE: amino acid                                            
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: Protein                                           
          (B) LOCATION: 1..102                                            
#/label= GENERIC-SEQ-6FORMATION:                                          
#AWHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM                         
#ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN                          
               THE SPECI - #FICATION"                                     
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                            
- Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xa - #a Xaa Xaa Gly Trp Xaa     
#                15                                                       
- Xaa Trp Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Al - #a Xaa Tyr Cys Xaa Gly     
#            30                                                           
- Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Asn His Ala     
#        45                                                               
- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa     
#    60                                                                   
- Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Leu Xaa Xaa     
#80                                                                       
- Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xa - #a Xaa Xaa Met Xaa Val     
#                95                                                       
- Xaa Xaa Cys Xaa Cys Xaa                                                 
            100                                                           
- (2) INFORMATION FOR SEQ ID NO:32:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#pairs    (A) LENGTH: 1247 base                                           
          (B) TYPE: nucleic acid                                          
          (C) STRANDEDNESS: single                                        
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: cDNA                                            
-     (ix) FEATURE:                                                       
          (A) NAME/KEY: CDS                                               
          (B) LOCATION: 84..1199                                          
#/product= "GDF-1"R INFORMATION:                                          
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                            
- GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CT - #GCGCACTC   
  60                                                                      
- TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CA - #A GGT CCC TGC       
 110                                                                      
#Gly Pro Cys Pro Pro Pro Gln Gln                                          
#       5  1                                                              
- GGC CAC CAC CTC CTC CTC CTC CTG GCC CTG CT - #G CTG CCC TCG CTG CCC     
 158                                                                      
Gly His His Leu Leu Leu Leu Leu Ala Leu Le - #u Leu Pro Ser Leu Pro       
# 25                                                                      
- CTG ACC CGC GCC CCC GTG CCC CCA GGC CCA GC - #C GCC GCC CTG CTC CAG     
 206                                                                      
Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Al - #a Ala Ala Leu Leu Gln       
#                 40                                                      
- GCT CTA GGA CTG CGC GAT GAG CCC CAG GGT GC - #C CCC AGG CTC CGG CCG     
 254                                                                      
Ala Leu Gly Leu Arg Asp Glu Pro Gln Gly Al - #a Pro Arg Leu Arg Pro       
#             55                                                          
- GTT CCC CCG GTC ATG TGG CGC CTG TTT CGA CG - #C CGG GAC CCC CAG GAG     
 302                                                                      
Val Pro Pro Val Met Trp Arg Leu Phe Arg Ar - #g Arg Asp Pro Gln Glu       
#         70                                                              
- ACC AGG TCT GGC TCG CGG CGG ACG TCC CCA GG - #G GTC ACC CTG CAA CCG     
 350                                                                      
Thr Arg Ser Gly Ser Arg Arg Thr Ser Pro Gl - #y Val Thr Leu Gln Pro       
#     85                                                                  
- TGC CAC GTG GAG GAG CTG GGG GTC GCC GGA AA - #C ATC GTG CGC CAC ATC     
 398                                                                      
Cys His Val Glu Glu Leu Gly Val Ala Gly As - #n Ile Val Arg His Ile       
#105                                                                      
- CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG GA - #G CCT GTC TCG GCC GCG     
 446                                                                      
Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser Gl - #u Pro Val Ser Ala Ala       
#               120                                                       
- GGG CAT TGC CCT GAG TGG ACA GTC GTC TTC GA - #C CTG TCG GCT GTG GAA     
 494                                                                      
Gly His Cys Pro Glu Trp Thr Val Val Phe As - #p Leu Ser Ala Val Glu       
#           135                                                           
- CCC GCT GAG CGC CCG AGC CGG GCC CGC CTG GA - #G CTG CGT TTC GCG GCG     
 542                                                                      
Pro Ala Glu Arg Pro Ser Arg Ala Arg Leu Gl - #u Leu Arg Phe Ala Ala       
#       150                                                               
- GCG GCG GCG GCA GCC CCG GAG GGC GGC TGG GA - #G CTG AGC GTG GCG CAA     
 590                                                                      
Ala Ala Ala Ala Ala Pro Glu Gly Gly Trp Gl - #u Leu Ser Val Ala Gln       
#   165                                                                   
- GCG GGC CAG GGC GCG GGC GCG GAC CCC GGG CC - #G GTG CTG CTC CGC CAG     
 638                                                                      
Ala Gly Gln Gly Ala Gly Ala Asp Pro Gly Pr - #o Val Leu Leu Arg Gln       
170                 1 - #75                 1 - #80                 1 -   
#85                                                                       
- TTG GTG CCC GCC CTG GGG CCG CCA GTG CGC GC - #G GAG CTG CTG GGC GCC     
 686                                                                      
Leu Val Pro Ala Leu Gly Pro Pro Val Arg Al - #a Glu Leu Leu Gly Ala       
#               200                                                       
- GCT TGG GCT CGC AAC GCC TCA TGG CCG CGC AG - #C CTC CGC CTG GCG CTG     
 734                                                                      
Ala Trp Ala Arg Asn Ala Ser Trp Pro Arg Se - #r Leu Arg Leu Ala Leu       
#           215                                                           
- GCG CTA CGC CCC CGG GCC CCT GCC GCC TGC GC - #G CGC CTG GCC GAG GCC     
 782                                                                      
Ala Leu Arg Pro Arg Ala Pro Ala Ala Cys Al - #a Arg Leu Ala Glu Ala       
#       230                                                               
- TCG CTG CTG CTG GTG ACC CTC GAC CCG CGC CT - #G TGC CAC CCC CTG GCC     
 830                                                                      
Ser Leu Leu Leu Val Thr Leu Asp Pro Arg Le - #u Cys His Pro Leu Ala       
#   245                                                                   
- CGG CCG CGG CGC GAC GCC GAA CCC GTG TTG GG - #C GGC GGC CCC GGG GGC     
 878                                                                      
Arg Pro Arg Arg Asp Ala Glu Pro Val Leu Gl - #y Gly Gly Pro Gly Gly       
250                 2 - #55                 2 - #60                 2 -   
#65                                                                       
- GCT TGT CGC GCG CGG CGG CTG TAC GTG AGC TT - #C CGC GAG GTG GGC TGG     
 926                                                                      
Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Ph - #e Arg Glu Val Gly Trp       
#               280                                                       
- CAC CGC TGG GTC ATC GCG CCG CGC GGC TTC CT - #G GCC AAC TAC TGC CAG     
 974                                                                      
His Arg Trp Val Ile Ala Pro Arg Gly Phe Le - #u Ala Asn Tyr Cys Gln       
#           295                                                           
- GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GG - #G TCC GGG GGG CCG CCG     
1022                                                                      
Gly Gln Cys Ala Leu Pro Val Ala Leu Ser Gl - #y Ser Gly Gly Pro Pro       
#       310                                                               
- GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC AT - #G CAC GCG GCC GCC CCG     
1070                                                                      
Ala Leu Asn His Ala Val Leu Arg Ala Leu Me - #t His Ala Ala Ala Pro       
#   325                                                                   
- GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC GC - #G CGC CTG TCG CCC ATC     
1118                                                                      
Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Al - #a Arg Leu Ser Pro Ile       
330                 3 - #35                 3 - #40                 3 -   
#45                                                                       
- TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC GT - #G GTG CTG CGG CAG TAT     
1166                                                                      
Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Va - #l Val Leu Arg Gln Tyr       
#               360                                                       
- GAG GAC ATG GTG GTG GAC GAG TGC GGC TGC CG - #C TAACCCGGGG CGGGCAGGGA   
1219                                                                      
Glu Asp Met Val Val Asp Glu Cys Gly Cys Ar - #g                           
#           370                                                           
#           1247   AATG CCGCGTGG                                          
- (2) INFORMATION FOR SEQ ID NO:33:                                       
-      (i) SEQUENCE CHARACTERISTICS:                                      
#acids    (A) LENGTH: 372 amino                                           
          (B) TYPE: amino acid                                            
          (D) TOPOLOGY: linear                                            
-     (ii) MOLECULE TYPE: protein                                         
-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                            
- Met Pro Pro Pro Gln Gln Gly Pro Cys Gly Hi - #s His Leu Leu Leu Leu     
#                 15                                                      
- Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Th - #r Arg Ala Pro Val Pro     
#             30                                                          
- Pro Gly Pro Ala Ala Ala Leu Leu Gln Ala Le - #u Gly Leu Arg Asp Glu     
#         45                                                              
- Pro Gln Gly Ala Pro Arg Leu Arg Pro Val Pr - #o Pro Val Met Trp Arg     
#     60                                                                  
- Leu Phe Arg Arg Arg Asp Pro Gln Glu Thr Ar - #g Ser Gly Ser Arg Arg     
# 80                                                                      
- Thr Ser Pro Gly Val Thr Leu Gln Pro Cys Hi - #s Val Glu Glu Leu Gly     
#                 95                                                      
- Val Ala Gly Asn Ile Val Arg His Ile Pro As - #p Arg Gly Ala Pro Thr     
#           110                                                           
- Arg Ala Ser Glu Pro Val Ser Ala Ala Gly Hi - #s Cys Pro Glu Trp Thr     
#       125                                                               
- Val Val Phe Asp Leu Ser Ala Val Glu Pro Al - #a Glu Arg Pro Ser Arg     
#   140                                                                   
- Ala Arg Leu Glu Leu Arg Phe Ala Ala Ala Al - #a Ala Ala Ala Pro Glu     
145                 1 - #50                 1 - #55                 1 -   
#60                                                                       
- Gly Gly Trp Glu Leu Ser Val Ala Gln Ala Gl - #y Gln Gly Ala Gly Ala     
#               175                                                       
- Asp Pro Gly Pro Val Leu Leu Arg Gln Leu Va - #l Pro Ala Leu Gly Pro     
#           190                                                           
- Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Tr - #p Ala Arg Asn Ala Ser     
#       205                                                               
- Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Le - #u Arg Pro Arg Ala Pro     
#   220                                                                   
- Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Le - #u Leu Leu Val Thr Leu     
225                 2 - #30                 2 - #35                 2 -   
#40                                                                       
- Asp Pro Arg Leu Cys His Pro Leu Ala Arg Pr - #o Arg Arg Asp Ala Glu     
#               255                                                       
- Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cy - #s Arg Ala Arg Arg Leu     
#           270                                                           
- Tyr Val Ser Phe Arg Glu Val Gly Trp His Ar - #g Trp Val Ile Ala Pro     
#       285                                                               
- Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gl - #n Cys Ala Leu Pro Val     
#   300                                                                   
- Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Le - #u Asn His Ala Val Leu     
305                 3 - #10                 3 - #15                 3 -   
#20                                                                       
- Arg Ala Leu Met His Ala Ala Ala Pro Gly Al - #a Ala Asp Leu Pro Cys     
#               335                                                       
- Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Va - #l Leu Phe Phe Asp Asn     
#           350                                                           
- Ser Asp Asn Val Val Leu Arg Gln Tyr Glu As - #p Met Val Val Asp Glu     
#       365                                                               
- Cys Gly Cys Arg                                                         
    370                                                                   
__________________________________________________________________________

Claims (22)

What is claimed is:
1. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system into the bloodstream, wherein it retains morphogenic activity, said morphogen comprising an acid-stable, protease-resistant dimeric protein that
(i) induces tissue-specific morphogenesis of at least mammalian bone tissue; and
(ii) comprises a pair of folded polypeptides, each having an amino acid sequence comprising a conserved cysteine skeleton of approximately 102 amino acids, wherein cysteines occupy relative positions 1, 30, 34, 66, 67, 99, and 101, and wherein at least 70% of amino acids in said skeleton are selected independently from amino acids occupying corresponding positions within the C-terminal seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5, and conservative substitution variants of said amino acids,
said morphogen being dispersed in said composition in a dosage sufficient to induce tissue-specific morphogenesis and to protect mammalian tissue from tissue damage or reduced tissue function due to metabolic stress or dysfunction.
2. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system into the bloodstream, wherein it retains morphogenic activity, said morphogen comprising an acid-stable, protease-resistant dimeric protein that
(i) induces tissue-specific morphogenesis of at least mammalian bone tissue; and
(ii) comprises a pair of folded polypeptides, each having an amino acid sequence comprising a conserved cysteine skeleton of approximately 102 amino acids, wherein cysteines occupy relative positions 1, 30, 34, 66, 67, 99, and 101, an wherein at least 70% of amino acids in said skeleton are selected independently from amino acids occupying corresponding positions within the C-terminal seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5, and conservative substitution variants said amino acids,
said morphogen being dispersed in said composition in a dosage sufficient to induce tissue-specific morphogenesis and to restore the function of senescent or quiescent mammalian tissue or mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress or dysfunction.
3. The composition of claim 1 or 2, wherein said morphogen is associated with a controlled release component, adapted such that the morphogen is released in a controlled manner in the gastrointestinal tract subsequent to passage through the stomach.
4. The composition of claim 1 or 2 adapted for enteral administration.
5. The composition of claim 1 or 2, formulated as a solid.
6. The composition of claim 5, wherein said solid is a tablet, troche or lozenge.
7. The composition of claim 1 or 2 formulated as a liquid.
8. The composition of claim 7, wherein said liquid is a beverage or a syrup.
9. The composition of claim 1 or 2, wherein said morphogen is associated with a morphogen-solubilizing molecule.
10. The composition of claim 9, wherein said molecule is casein or a derivative, salt or analog thereof.
11. The composition of claim 9, wherein said morphogen-solubilizing molecule comprises one or more prodomains selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, BMP6, and conservative substitution variants of any of the foregoing which retain morphogenic activity.
12. The composition of claim 1 or 2, wherein at least 80% of amino acids within said skeleton are selected independently from amino acids occupying corresponding positions within the C-terminal seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5, and conservative substitution variants of said amino acids.
13. The composition of claim 1 or 2, wherein said skeleton has greater than 60% amino acid sequence identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5.
14. The composition of claim 13, wherein said skeleton has greater than 65% amino acid sequence identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of Seq. ID No. 5.
15. The composition of claim 14, wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, each comprising a C-terminal seven cysteine domain of human OP-1, residues 43-139 of Seq. ID No. 5.
16. The composition of claim 1 or 2, wherein said nutrient is selected from the group consisting of proteins, amino acids, carbohydrates, lipids, fatty acids, sugars, nucleosides, and nucleotides.
17. The composition of claims 1 or 2, wherein said mineral is selected from the group consisting of calcium, phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese, and iodine.
18. The composition of claim 1 or 2, wherein said vitamin is selected from the group consisting of vitamin A, vitamin D3, vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E, vitamin K1, folic acid and biotin.
19. The composition of claim 1 or 2, wherein said morphogen is present in its pro form.
20. The composition of claim 19, wherein said morphogen is an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, each comprising a sequence of human pro OP-1, residues 30-431 of Seq. ID No. 17.
21. A composition comprising:
(a) a nutrient, trace mineral, or vitamin required for normal metabolism in mammalian tissue; and
(b) a morphogen capable of being absorbed from the digestive system into the bloodstream, wherein it retains morphogenic activity, said morphogen comprising an acid-stable, protease-resistant dimeric protein that
(i) induces tissue-specific morphogenesis of at least mammalian bone tissue; and
(ii) comprises a pair of folded polypeptides, each comprising an amino acid sequence defined by Generic Sequence 6, Seq. ID No. 31, said morphogen being dispersed in said composition at a dosage sufficient to induces tissue-specific morphogenesis and to protect mammalian tissue at risk of incurring tissue damage or reduced tissue function, or for treating mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress or dysfunction.
22. The composition of claim 21, wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, each comprising an amino acid sequence defined by OPX, Seq. ID No. 29.
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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030105004A1 (en) * 1991-03-11 2003-06-05 Curis, Inc. Novel morphogenic protein compositions of matter
US20030144229A1 (en) * 2000-01-14 2003-07-31 The Government Of The U.S.A., The Secretary Of The Department Of Health And Human Services Multiple CpG oligodeoxynucleotides and their use to induce an immune response
US20030224996A1 (en) * 1988-04-08 2003-12-04 Stryker Corporation Osteogenic devices
US20040077546A1 (en) * 1988-04-08 2004-04-22 Stryker Corporation Osteogenic proteins
US20040105872A1 (en) * 2002-09-18 2004-06-03 The Government Of The Usa As Represented By The Secretary Of The Dept. Of Health & Human Services Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides
US20040142469A1 (en) * 1994-07-15 2004-07-22 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US20040162240A1 (en) * 1999-04-16 2004-08-19 Mayo Foundation For Medical Education And Research Cobalamin conjugates useful as antitumor agents
US20050026245A1 (en) * 2001-12-20 2005-02-03 Klinman Dennis M. Use of cpg oligodeoxynucleotides to induce angiogenesis
US20050054825A1 (en) * 1988-04-08 2005-03-10 Stryker Biotech Corporation Osteogenic devices
EP1181035B1 (en) * 1999-05-28 2005-03-30 Novartis AG Substantially oil-free cyclosporin compositions
US20050143340A1 (en) * 2001-06-20 2005-06-30 Collins Douglas A. Adenosyl-cobalamin fortified compositions
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
US20060166862A1 (en) * 2000-10-25 2006-07-27 Collins Douglas A Transcobalamin receptor binding conjugates useful for treating abnormal cellular proliferation
US20060240062A1 (en) * 2002-09-10 2006-10-26 Klaus Hellerbrand Metal implant coated under reduced oxygen concentration with osteoinductive protein
US7147839B2 (en) 1998-05-29 2006-12-12 Curis, Inc. Methods for evaluating tissue morphogenesis and activity
US20070265187A1 (en) * 2004-04-29 2007-11-15 Slobodan Vukicevic Oral Formulations Comprising Bone Morphogenetic Proteins For Treating Metabolic Bone Diseases
US7354909B2 (en) 2001-08-14 2008-04-08 The United States Of America As Represented By Secretary Of The Department Of Health And Human Services Method for rapid generation of mature dendritic cells
US20080226745A1 (en) * 2001-08-24 2008-09-18 Martek Biosciences Corporation Products Containing Highly Unsaturated Fatty Acids for Use by Women During Stages of Preconception, Pregnancy and Lactation/Post-Partum
US20080233170A1 (en) * 1992-02-21 2008-09-25 Stryker Corporation Osteogenic Proteins
EP1988395A1 (en) 1997-05-30 2008-11-05 Curis, Inc. Methods for evaluating tissue morphogenesis and morphogenic activity
US7462345B2 (en) 1995-11-13 2008-12-09 Mayo Foundation For Medical Education And Research Radionuclide labeling of vitamin B12 and coenzymes thereof
US7666674B2 (en) 2001-07-27 2010-02-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo
US20130064940A1 (en) * 2005-09-20 2013-03-14 Prolacta Bioscience, Inc. Methods for testing milk
US8466116B2 (en) 2001-12-20 2013-06-18 The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce epithelial cell growth
US8546334B2 (en) 2001-11-19 2013-10-01 Scil Technology Gmbh Device having osteoinductive and osteoconductive properties
WO2014208868A1 (en) * 2013-06-24 2014-12-31 고려대학교산학협력단 Composition for preventing or treating psoriasis, containing bmp6, and dietary supplement for preventing or alleviating psoriasis, containing bmp6

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3626069A (en) * 1970-12-30 1971-12-07 Merck & Co Inc Growth promotion
US4177293A (en) * 1978-11-15 1979-12-04 Mlekarensky Prumysl, Generalni Reditelstvi Process of manufacture of a butter spread
US4418091A (en) * 1979-10-17 1983-11-29 Centrale Veevoederfabriek "Sloten" Process for preparing a dry, powdery milk product
US4440860A (en) * 1980-01-18 1984-04-03 The Children's Medical Center Corporation Stimulating cell growth
US4466958A (en) * 1981-03-18 1984-08-21 Morrison L M Food supplement
EP0148155A2 (en) * 1984-01-04 1985-07-10 International Genetic Engineering, Inc. (Ingene) Osteogenic factors
WO1988000205A1 (en) * 1986-07-01 1988-01-14 Genetics Institute, Inc. Novel osteoinductive compositions
EP0295009A2 (en) * 1987-06-03 1988-12-14 Baylor College Of Medicine Lactoferrin as a dietary ingredient promoting the growth of the gastrointestinal tract
EP0313515A1 (en) * 1987-10-01 1989-04-26 Ciba-Geigy Ag A polypeptide growth factor from milk
WO1989009788A2 (en) * 1988-04-08 1989-10-19 Creative Biomolecules, Inc. Biosynthetic osteogenic proteins and osteogenic devices containing them
US4877864A (en) * 1987-03-26 1989-10-31 Genetics Institute, Inc. Osteoinductive factors
WO1989010409A1 (en) * 1988-04-08 1989-11-02 Genetics Institute, Inc. Bone and cartilage inductive compositions
WO1990003733A1 (en) * 1988-10-11 1990-04-19 International Genetic Engineering, Inc. Osteogenic factors
US4968590A (en) * 1988-04-08 1990-11-06 Stryker Corporation Osteogenic proteins and polypeptides
US4975526A (en) * 1989-02-23 1990-12-04 Creative Biomolecules, Inc. Bone collagen matrix for zenogenic implants
US4994442A (en) * 1987-05-29 1991-02-19 Union Industrial Y Agro-Ganadera, S.A. (Uniasa) Method for stimulation or repair and regeneration of intestinal gut cells in infants and enhancing the immune response of t-cells
EP0416578A2 (en) * 1989-09-06 1991-03-13 Takeda Chemical Industries, Ltd. Protein, DNA and use thereof
US5008240A (en) * 1985-08-06 1991-04-16 Collagen Corporation Method of treating inflammation
US5011691A (en) * 1988-08-15 1991-04-30 Stryker Corporation Osteogenic devices
US5013649A (en) * 1986-07-01 1991-05-07 Genetics Institute, Inc. DNA sequences encoding osteoinductive products
US5013569A (en) * 1990-05-21 1991-05-07 Century Laboratories, Inc. Infant formula
US5108989A (en) * 1990-04-04 1992-04-28 Genentech, Inc. Method of predisposing mammals to accelerated tissue repair
US5108753A (en) * 1988-04-08 1992-04-28 Creative Biomolecules Osteogenic devices
US5141905A (en) * 1986-07-01 1992-08-25 Rosen Vicki A Dna sequences encoding bmp-7 proteins
US5169666A (en) * 1991-11-14 1992-12-08 The United States Of America As Represented By The Secretary Of Agriculture Preparation of simulated human milk protein by low temperature microfiltration
US5221734A (en) * 1987-10-01 1993-06-22 Ciba-Geigy Corporation Process for preparing a polypeptide growth factor for milk

Patent Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3626069A (en) * 1970-12-30 1971-12-07 Merck & Co Inc Growth promotion
US4177293A (en) * 1978-11-15 1979-12-04 Mlekarensky Prumysl, Generalni Reditelstvi Process of manufacture of a butter spread
US4418091A (en) * 1979-10-17 1983-11-29 Centrale Veevoederfabriek "Sloten" Process for preparing a dry, powdery milk product
US4440860A (en) * 1980-01-18 1984-04-03 The Children's Medical Center Corporation Stimulating cell growth
US4466958A (en) * 1981-03-18 1984-08-21 Morrison L M Food supplement
EP0148155A2 (en) * 1984-01-04 1985-07-10 International Genetic Engineering, Inc. (Ingene) Osteogenic factors
US5008240A (en) * 1985-08-06 1991-04-16 Collagen Corporation Method of treating inflammation
WO1988000205A1 (en) * 1986-07-01 1988-01-14 Genetics Institute, Inc. Novel osteoinductive compositions
US5013649A (en) * 1986-07-01 1991-05-07 Genetics Institute, Inc. DNA sequences encoding osteoinductive products
US5141905A (en) * 1986-07-01 1992-08-25 Rosen Vicki A Dna sequences encoding bmp-7 proteins
US4877864A (en) * 1987-03-26 1989-10-31 Genetics Institute, Inc. Osteoinductive factors
US5066500A (en) * 1987-05-29 1991-11-19 Union Industrial Y Agro-Ganadera, S. A. (Uniasa) Infant formulas and nutrition products enriched with nucleosides and/or nucleotides and processes for their preparation
US4994442A (en) * 1987-05-29 1991-02-19 Union Industrial Y Agro-Ganadera, S.A. (Uniasa) Method for stimulation or repair and regeneration of intestinal gut cells in infants and enhancing the immune response of t-cells
EP0295009A2 (en) * 1987-06-03 1988-12-14 Baylor College Of Medicine Lactoferrin as a dietary ingredient promoting the growth of the gastrointestinal tract
US5221734A (en) * 1987-10-01 1993-06-22 Ciba-Geigy Corporation Process for preparing a polypeptide growth factor for milk
EP0313515A1 (en) * 1987-10-01 1989-04-26 Ciba-Geigy Ag A polypeptide growth factor from milk
US4968590A (en) * 1988-04-08 1990-11-06 Stryker Corporation Osteogenic proteins and polypeptides
WO1989010409A1 (en) * 1988-04-08 1989-11-02 Genetics Institute, Inc. Bone and cartilage inductive compositions
US5108753A (en) * 1988-04-08 1992-04-28 Creative Biomolecules Osteogenic devices
WO1989009787A2 (en) * 1988-04-08 1989-10-19 Creative Biomolecules, Inc. Osteogenic devices
WO1989009788A2 (en) * 1988-04-08 1989-10-19 Creative Biomolecules, Inc. Biosynthetic osteogenic proteins and osteogenic devices containing them
US5011691A (en) * 1988-08-15 1991-04-30 Stryker Corporation Osteogenic devices
WO1990003733A1 (en) * 1988-10-11 1990-04-19 International Genetic Engineering, Inc. Osteogenic factors
US5106626A (en) * 1988-10-11 1992-04-21 International Genetic Engineering, Inc. Osteogenic factors
US4975526A (en) * 1989-02-23 1990-12-04 Creative Biomolecules, Inc. Bone collagen matrix for zenogenic implants
EP0416578A2 (en) * 1989-09-06 1991-03-13 Takeda Chemical Industries, Ltd. Protein, DNA and use thereof
US5108989A (en) * 1990-04-04 1992-04-28 Genentech, Inc. Method of predisposing mammals to accelerated tissue repair
US5013569A (en) * 1990-05-21 1991-05-07 Century Laboratories, Inc. Infant formula
US5169666A (en) * 1991-11-14 1992-12-08 The United States Of America As Represented By The Secretary Of Agriculture Preparation of simulated human milk protein by low temperature microfiltration

Non-Patent Citations (109)

* Cited by examiner, † Cited by third party
Title
AAP Committee on Nutrition, Pediatrics , 75:976 986 (1985). *
AAP Committee on Nutrition, Pediatrics, 75:976-986 (1985).
Behringer et al., Nature , 345:167 170 (1990). *
Behringer et al., Nature, 345:167-170 (1990).
Berkow et al, The Merck Manual , 14 th ed., pp. 869 889 (1982). *
Berkow et al, The Merck Manual, 14th ed., pp. 869-889 (1982).
Bowie et al. 1990. Science 247:1306 1310. *
Bowie et al. 1990. Science 247:1306-1310.
Cate et al., Cell , 45:685 698 (1986). *
Cate et al., Cell, 45:685-698 (1986).
Celeste et al., PNAS , 87:9843 9847 (1990). *
Celeste et al., PNAS, 87:9843-9847 (1990).
Chomczyaski et al., Anal. Bioch. , 162:156 159 (1987). *
Chomczyaski et al., Anal. Bioch., 162:156-159 (1987).
D Allesandro et al. (1991), Abstract Q 105 J. Cell. Biochem . *
D'Allesandro et al. (1991), Abstract Q-105 J. Cell. Biochem.
Edelman, Ann. Rev. Cell. Biol. , 2:81 116 (1986). *
Edelman, Ann. Rev. Cell. Biol., 2:81-116 (1986).
Engvall, Methods in Enzymology , 70:419 439 (1980). *
Engvall, Methods in Enzymology, 70:419-439 (1980).
ESPGAN Committee of Nutrition, Acta Paed. Scan. Suppl. , 330:7 27 (1987). *
ESPGAN Committee of Nutrition, Acta Paed. Scan. Suppl., 330:7-27 (1987).
Gennaro (1990) Remington s Pharmaceutical Sciences (Mack Publishers, N.Y.). *
Gennaro (1990) Remington's Pharmaceutical Sciences (Mack Publishers, N.Y.).
Green et al., Nature , 347:391 394 (1990). *
Green et al., Nature, 347:391-394 (1990).
Israel et al. (1991), Abstract Q 111 J. Cell. Biochem . *
Israel et al. (1991), Abstract Q-111 J. Cell. Biochem.
Israel et al. (1992), "Expression and Characterization of Bone Morphogenetic Protein-2 in Chinese Hamster Ovary Cells," 7 Growth Factors 139-150.
Israel et al. (1992), Expression and Characterization of Bone Morphogenetic Protein 2 in Chinese Hamster Ovary Cells, 7 Growth Factors 139 150. *
Jin et al., J. Protein Chemistry , 10:565 575 (1991). *
Jin et al., J. Protein Chemistry, 10:565-575 (1991).
Katagiri et al. (1990), "The Non-Osteogenic Mouse Pluripotent Cell Line, C3H10T1/2 is Induced to Differentiate Into Osteoblastic Cells by Recombinant Human Bone Morphogenetic Protein-2," 172 Biochem. Biophys. Res. 1:295-299.
Katagiri et al. (1990), The Non Osteogenic Mouse Pluripotent Cell Line, C3H10T1/2 is Induced to Differentiate Into Osteoblastic Cells by Recombinant Human Bone Morphogenetic Protein 2, 172 Biochem. Biophys. Res. 1:295 299. *
Kuberasampath et al., J. Biol. Chem. , 265:13198 13205 (1990). *
Kuberasampath et al., J. Biol. Chem., 265:13198-13205 (1990).
Lee, Molecular Endocrinology , 4:1034 1040 (1990). *
Lee, Molecular Endocrinology, 4:1034-1040 (1990).
Lee, PNAS , 88:4250 4254 (1991). *
Lee, PNAS, 88:4250-4254 (1991).
Lefer et al. (1990) Science, vol. 249, pp. 61 64. *
Lefer et al. (1990) Science, vol. 249, pp. 61-64.
Letterio et al. Science 264:1936 1938, 1994. *
Letterio et al. Science 264:1936-1938, 1994.
Lyons et al., Genes & Development , 3:1657 1668 (1989). *
Lyons et al., Genes & Development, 3:1657-1668 (1989).
Lyons et al., PNAS , 86:4554 4558 (1989). *
Lyons et al., PNAS, 86:4554-4558 (1989).
Mason et al., Mol. Endocrinology , 3:1352 1358 (1989). *
Mason et al., Mol. Endocrinology, 3:1352-1358 (1989).
Mason et al., Nature , 318:659 663 (1985). *
Mason et al., Nature, 318:659-663 (1985).
Massagu e (1987) Cell, vol. 49, pp. 437 438. *
Massague (1987) Cell, vol. 49, pp. 437-438.
Miller et al., Cancer Research , 42:2589 3594 (1987). *
Miller et al., Cancer Research, 42:2589-3594 (1987).
Ozkaynak et al., Biochem. Biophys. Res. Comm. , 179:116 123 (1991). *
Ozkaynak et al., Biochem. Biophys. Res. Comm., 179:116-123 (1991).
Ozkaynak et al., Embo J. , 9:2085 2093 (1990). *
Ozkaynak et al., Embo J., 9:2085-2093 (1990).
Padgett et al. (1993), "Human BMP Sequences Can Confer Normal Dorsal-Ventral Patterning in the Drosophila Embryo," 90 PNAS 2905-2909.
Padgett et al. (1993), Human BMP Sequences Can Confer Normal Dorsal Ventral Patterning in the Drosophila Embryo, 90 PNAS 2905 2909. *
Padgett et al., Nature , 325:81 84 (1987). *
Padgett et al., Nature, 325:81-84 (1987).
Panganiban et al., Mol. and Cell. Biol. , 10:2669 2677 (1990). *
Panganiban et al., Mol. and Cell. Biol., 10:2669-2677 (1990).
Pepinsky et al. (1988) Journal of Biological Chemistry, vol. 263, pp. 18961 18964. *
Pepinsky et al. (1988) Journal of Biological Chemistry, vol. 263, pp. 18961-18964.
Reinhardt. Annals od Allergy 53:597 601, 1984. *
Reinhardt. Annals od Allergy 53:597-601, 1984.
Rogers et al. (1992), "Bone Morphogenetic Proteins-2 and -4 are Involved In The Retinoic Acid-Induced Differentiation of Embryonal Carcinoma Cells," 3 Mol. Biol. Cell 2:189-196.
Rogers et al. (1992), Bone Morphogenetic Proteins 2 and 4 are Involved In The Retinoic Acid Induced Differentiation of Embryonal Carcinoma Cells, 3 Mol. Biol. Cell 2:189 196. *
Rosen et al. (1989), "Purification and Molecular Cloning of a Novel Group of BMPS and Localization of BMP MRNA In Developing Bone," 20 Connect. Tissue Res. 1-4:313-319.
Rosen et al. (1989), Purification and Molecular Cloning of a Novel Group of BMPS and Localization of BMP MRNA In Developing Bone, 20 Connect. Tissue Res. 1 4:313 319. *
Rosen et al.; Celeste et al. (1990), 2 Abstracts J. Cell Biochem. Suppl. 14 Part E, 004:33, 54:105. *
Rosen et al.; Wang et al.; and Wozney et al. (1988), 3 Abstracts Calc. Tissue Int. 42 (Suppl.): A35(136), A37(146,147). *
Sampath et al., PNAS , 80:6591 6595 (1983). *
Sampath et al., PNAS, 80:6591-6595 (1983).
Schubert et al., Nature , 344:868 870 (1990). *
Schubert et al., Nature, 344:868-870 (1990).
Smith et al., Nature , 345:729 731 (1990). *
Smith et al., Nature, 345:729-731 (1990).
Sokol et al., Science , 249:561 563 (1990). *
Sokol et al., Science, 249:561-563 (1990).
Takuwa et al. (1991), "Bone Morphogenetic Protein-2 Stimulates Alkaline Phosphatase Activity and Collagen Synthesis in Cultured Osteoblastic Cells, MC3T3-E1," 174 Biochem. Biophys. Res. Comm. 1:96-101.
Takuwa et al. (1991), Bone Morphogenetic Protein 2 Stimulates Alkaline Phosphatase Activity and Collagen Synthesis in Cultured Osteoblastic Cells, MC3T3 E1, 174 Biochem. Biophys. Res. Comm. 1:96 101. *
Thies et al. (1992), "Recombinant Human Bone Morphogenetic Protein-2 Induces Osteoblastic Differentiation in W-20-17 Stromal Cells," 130 Endocrinology 3:1318-1324.
Thies et al. (1992), Recombinant Human Bone Morphogenetic Protein 2 Induces Osteoblastic Differentiation in W 20 17 Stromal Cells, 130 Endocrinology 3:1318 1324. *
Wang et al., PNAS , 85:9484 9488 (1988). *
Wang et al., PNAS , 87:2220 2224 (1990). *
Wang et al., PNAS, 85:9484-9488 (1988).
Wang et al., PNAS, 87:2220-2224 (1990).
Weeks, Cell , 51:861 867 (1987). *
Weeks, Cell, 51:861-867 (1987).
Wharton et al., PNAS , 88:9214 9218 (1991). *
Wharton et al., PNAS, 88:9214-9218 (1991).
Wozney et al. (1990), "Bone Morphogenic Proteins," 1 Prog. In Growth Factor Res. 267-280.
Wozney et al. (1990), "Growth Factors Influencing Bone Development," 13 J. Cell Sci. Suppl. 149-156.
Wozney et al. (1990), Bone Morphogenic Proteins, 1 Prog. In Growth Factor Res. 267 280. *
Wozney et al. (1990), Growth Factors Influencing Bone Development, 13 J. Cell Sci. Suppl. 149 156. *
Wozney et al. (1992), "The Bone Morphogenetic Protein Family and Osteogenesis," 32 Mol. Reprod. Dev. 2:160-167.
Wozney et al. (1992), The Bone Morphogenetic Protein Family and Osteogenesis, 32 Mol. Reprod. Dev. 2:160 167. *
Wozney et al., Science , 242:1528 1534 (1988). *
Wozney et al., Science, 242:1528-1534 (1988).
Wozney et al.; Celeste et al.; Rosen et al. (1992), Abstracts 76(WO26), 100(W502) and 103(W513) J. Cell Biochem. Suppl. 16 Part F. *
Yamaguchi et al. (1991), "Recombinant Human Bone Morphogenetic Protein-2 Stimulates Osteoblastic Maturation and Inhibits Myogenic Differentiation In Vitro," 113 J. Cell Biol. 3:681-687.
Yamaguchi et al. (1991), Recombinant Human Bone Morphogenetic Protein 2 Stimulates Osteoblastic Maturation and Inhibits Myogenic Differentiation In Vitro, 113 J. Cell Biol. 3:681 687. *
Yannas, Angew. Chem. Int. Ed. Engl. , 29:20 35 (1990). *
Yannas, Angew. Chem. Int. Ed. Engl., 29:20-35 (1990).

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US20040152656A1 (en) * 1994-07-15 2004-08-05 University Of Iowa Research Foundation Immunomodulatory oligonucleotides
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